Macrophage Colony Research Articles

Modification of intrinsic ovarian cancer tumor factors by Wnt-pathway agents influences macrophage recruitment and activity.

e14514 Background: The Wnt/B-catenin pathway is associated with a “cold” phenotype in the gynecologic malignancy tumor microenvironment (TME), resulting in immunosuppression. Research is limited regarding this pathway’s effects on TME myeloid cell populations. We aimed to characterize the impact of Wnt pathway modulation on macrophage activity in ovarian cancer. Methods: Syngeneic murine models harboring ID8 parental (ID8par) tumors were treated with DKN-01 and CGX-1321 as single agents or in sequence (DKN-01 followed by CGX-1321.) Flow cytometry analyses were performed on harvested omenta and blood. Ascites from 10 high grade serous epithelial ovarian cancer patients were treated with DKN-01 (Wnt activator) or CGX-1321 (Wnt inhibitor) for 48 hours; multiplex cytokine array was performed to determine changes in cytokine/chemokine expression. Lastly, we performed co-culture analyses on the effects of ID8par and ID8p53-/- isolated cell media on murine macrophage (RAW264.7) activity. Results: In the ID8par model, sequential DKN-01/CGX-1321 resulted in the greatest macrophage influx into the TME. CGX-1321 monotherapy increased the M2:M1 macrophage ratio (pro-tumor) while DKN-01 monotherapy resulted in decreased M2:M1 ratio (anti-tumor.) In human ascites, DKN-01 increased macrophage colony stimulating factor (M-CSF) expression. Of note, ascites from a patient harboring a p53 mutation demonstrated an increase in M1 polarization cytokines IFNy and TNFa in response to DKN-01; CGX-1321 treatment resulted in increased M2 polarization cytokines IL-4 and IL-13. In co-culture analysis, ID8par isolated media increased RAW264.7 migration and polarization towards an M2 phenotype; ID8p53-/- isolated media polarized towards an M1 phenotype. Conclusions: Macrophage activity influences immune responses and tumor behavior. For example, M2 macrophages are pro-tumor and can enhance tumor growth and immune evasion. In our studies, we demonstrated that macrophage recruitment and polarization (M1 vs M2) are influenced by genetic alterations that can be modified by treatment. The ID8par model positively influenced M2 macrophage activity; ID8p53-/- cells positively influenced M1 macrophage activity. Importantly, DKN-01 treatment reverses the M2-dominant phenotype in ID8par model towards M1. These results suggest that p53 mutation and/or DKN-01 treatment in ovarian cancer influences M1 anti-tumor macrophage activity, and posits a novel targetable pathway. Further studies should investigate Wnt/B-catenin modulation on macrophage activity for immunotherapy in gynecologic malignancies.

OP0059 MAVRILIMUMAB (ANTI GM-CSF RECEPTOR Α MONOCLONAL ANTIBODY) REDUCES RISK OF FLARE AND INCREASES SUSTAINED REMISSION IN A PHASE 2 TRIAL OF PATIENTS WITH GIANT CELL ARTERITIS

Background:T helper (Th)1 and Th17 lymphocytes play a role in the pathogenesis of giant cell arteritis (GCA). Current treatments primarily target the Th17 axis, possibly leaving residual Th1 activity. Granulocyte macrophage colony stimulating factor (GM-CSF), a mediator of Th1 and Th17 cells, is a pathogenic factor in GCA.Objectives:To evaluate the efficacy and safety of the GM-CSF inhibitor mavrilimumab in patients with GCA.Methods:Randomized, double-blind, placebo-controlled phase 2 trial enrolling patients with active, biopsy- or imaging-proven new onset (N/O) or relapsing refractory (R/R) GCA. Active disease: GCA symptoms and erythrocyte sedimentation rate (ESR) (>30 mm/hr) and/or C-reactive protein (CRP) (≥1 mg/dL) elevation within 6 weeks from randomization. Corticosteroid-induced remission (resolution of GCA symptoms and CRP <1 mg/dL or ESR <20 mm/hr) was required by baseline. 3:2 randomization to mavrilimumab 150 mg or placebo subcutaneously every 2 weeks and protocol-defined 26-week prednisone taper starting at 20-60 mg/day.Primary efficacy endpoint: time to first adjudicated flare (ESR ≥30 mm/hr and/or CRP ≥1 mg/dL and GCA symptoms or new/worsening vasculitis on imaging) by Week 26 in all treated patients. Key secondary endpoint: sustained remission through Week 26. Safety up to Week 38 was assessed.Results:70 patients (35 N/O, 35 R/R) were enrolled (mavrilimumab [N=42] or placebo [N=28]). Mean (SD) age was 69.7 (7.48) years and 71.4% were female. Flare by Week 26 occurred in 8 (19%) and 13 (46.4%) patients receiving mavrilimumab and placebo, respectively (27.4 percentage points reduction). Median time to flare by Week 26 could not be estimated in the mavrilimumab group due to too few events (Not Estimable) and was 25.1 weeks [95% CI: (16.0, NE)] in the placebo group (HR [95% CI] 0.38 [0.15, 0.92]; p=0.0263) (Figure). Sustained remission at Week 26 occurred in 83.2% of patients receiving mavrilimumab and 49.9% of those receiving placebo (33.4 percentage points increase; p=0.0038). Results were consistent across disease type subgroups (HR for flare: N/O 0.29 [95% CI: 0.06, 1.31; nominal p= 0.0873]; R/R 0.43 [95% CI: 0.14, 1.30]; nominal p=0.1231), although not powered for significance (Table). Adverse events (AEs), mostly mild to moderate, were comparable between groups. There were 5 serious AEs (mavrilimumab 2 [4.8%], placebo 3 [10.7%]), none drug-related. No deaths or vision loss occurred. No adjudicated cases of pulmonary alveolar proteinosis were observed.Table 1.Efficacy at Week 26All Patients [1]SubgroupsN/OR/RMavrilimu-mab (N=42)Placebo (N=28)Mavrilimu-mab (N=24)Placebo (N=11)Mavrilimu-mab (N=18)Placebo (N=17)Patients with Flare, n (%)8 (19.0)13 (46.4)3 (12.5)4 (36.4)5 (27.8)9 (52.9)Time to Flare (weeks) [2]Median, 95% CINE (NE, NE)25.1 (16.0, NE)NE (NE, NE)NE (11.7, NE)NE (16.4, NE)22.6 (16.0, NE)HR (Mavrilimumab vs Placebo), 95% CI [3]0.38 (0.15, 0.92)0.29 (0.06, 1.31)0.43 (0.14, 1.30)P-value [4] [5]0.02630.08730.1231Sustained Remission (%), 95% CI [6]83.2 (67.9, 91.6)49.9 (29.6, 67.3)91.3 (69.3, 97.7)62.3 (27.7, 84.0)72.2 (45.6, 87.4)41.7 (17.4, 64.5)Difference in Proportions (95% CI) [7]33.3 (10.7, 55.8)28.9 (-2.7, 60.5)30.6 (-2.1, 63.2)P-value [5] [7]0.00380.07270.0668NE = Not estimable. [1] Total mITT population. Stratified by randomization strata. [2] Kaplan-Meier. [3] Cox proportional-hazards model; treatment as covariate. [4] Log-rank test. [5] N/O and R/R subgroups not powered for significance; nominal p values reported. [6] Kaplan-Meier Survival Estimates with standard error. [7] Two-sided p-value for the difference in sustained remission between 2 arms using normal approximation. Placebo arm is reference.Conclusion:Mavrilimumab was superior to placebo on the primary and secondary efficacy endpoints of time to flare and sustained remission at week 26 in patients with GCA. Mavrilimumab was well tolerated, and no new safety signals were observed.Disclosure of Interests:Maria C. Cid Speakers bureau: meeting attendance support from Roche and Kiniksa, Paid instructor for: educational from GSK and Vifor, Consultant of: consulting for Janssen, GSK, and Abbvie, Grant/research support from: research grant from Kiniksa, Sebastian Unizony Consultant of: consulting for Janssen and Kiniksa, Grant/research support from: research support from Genentech, Lara Pupim Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, Fang Fang Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, Joseph Pirrello Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, Ai Ren Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, Manoj Samant Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, Teresa Zhou Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals, John F. Paolini Shareholder of: Kiniksa Pharmaceuticals, Employee of: Kiniksa Pharmaceuticals

TGFβ1, MMPs and cytokines profiles in ocular surface: Possible tear biomarkers for pseudoexfoliation.

Pseudoexfoliation (PXF) is a unique form of glaucoma characterized by accumulation of exfoliative material in the eyes. Changes in tear profile in disease stages may give us insights into molecular mechanisms involved in causing glaucoma in the eye. All patients were categorized into three main categories; pseudoexfoliation (PXF), pseudoexfoliation glaucoma (PXG) and cataract, which served as control. Cytokines, transforming growth factor β1 (TGFβ1), matrix metalloproteases (MMPs) and fibronectin (FN1) were assessed with multiplex bead assay, enzyme-linked immunosorbent assay (ELISA), gelatin zymography, and immunohistochemistry (IHC) respectively in different ocular tissues such as tears, tenon's capsule, aqueous humor (AH) and serum samples of patients with PXF stages. We found that TGFβ1, MMP-9 and FN1 protein expression were upregulated in tears, tenon's capsule and AH samples in PXG compared to PXF, though the MMP-9 protein activity was downregulated in PXG compared with control or PXF. We have also found that in PXG tears sample the fold change of TGF-α (Transforming Growth Factor-α), MDC (Macrophage Derived Chemokine), IL-8 (Interleukin-8), VEGF (Vascular Endothelial Growth Factor) were significantly downregulated and the levels of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), IP-10 (Interferon- γ produced protein-10) were significant upregulated. While in AH; IL-6 (Interleukin-6), IL-8, VEGF, IFN-a2 (Interferon- α2), GRO (Growth regulated alpha protein) levels were found lower and IL1a (Interleukin-1α) level was higher in PXG compared to PXF. And in serum; IFN-a2, Eotaxin, GM-CSF, Fractalkine, IL-10 (Interleukin-10), IL1Ra (Interleukin-1 receptor antagonist), IL-7 (Interleukin-7), IL-8, MIP1β (Macrophage Inflammatory Protein-1β), MCP-1 (Monocyte Chemoattractant Protein-1) levels were significantly upregulated and PDGF-AA (Platelet Derived Growth Factor-AA) level was downregulated in the patients with PXG compared to PXF. Altered expression of these molecules in tears may therefore be used as a signal for onset of glaucoma or for identifying eyes at risk of developing glaucoma in PXF.

Interleukin-35 Is Involved in Angiogenesis/Bone Remodeling Coupling Through T Helper 17/Interleukin-17 Axis.

ObjectiveOsteoporosis is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the suppressive role of interleukin (IL)-35 in nuclear factor kappa-B ligand receptor activator (RANKL) and macrophage colony stimulating factor (M-CSF)-induced osteoclastogenesis and angiogenesis in osteoclasts.MethodsOsteoclasts differentiation were induced by incubation of mouse leukemic monocyte/macrophage cell line RAW264.7 cells in the presence of RANKL and M-CSF and was assessed with tartrate-resistant acid phosphatase (TRAP) staining assay. The viability and apoptosis of RAW264.7 was measured using CCK-8 assay and flow cytometry, respectively. The expression of angiogenic genes and proteins were measured using RT-PCR, Western blots and ELISA. The inhibition of Th17/IL-17 axis was examined using plumbagin, which was demonstrated as an IL-17A related signaling pathway inhibitor.ResultsIL-35 inhibited the viability of RAW264.7 cells and promoted the apoptosis of RAW264.7 cells in a dose-dependent manner. Furthermore, IL-35 dose-dependently suppressed the expression of angiogenic markers including VEGF and its receptor. The suppressive effect of IL-35 was confirmed through the activation of Th17/IL-17 axis.ConclusionsWe demonstrated for the first time the immuno-suppressive function of IL-35 on RANKL and M-CSF-induced osteoclastogenesis and angiogenesis through Th17/IL-17 axis. Therapeutic approach involving augmentation of IL-35 regulatory response may serve as a novel treatment option for osteoporosis, especially by suppressing bone resorption and angiogenesis.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

MiR-128 Participates in the Pathogenesis of Slow Transit Constipation by Regulating the P38α/M-CSF Inflammatory Signaling Pathway

Background: Slow transit constipation (STC) is a gastrointestinal disorder that adversely affects the quality of life. MicroRNAs are involved in the pathogenesis of functional gastrointestinal disorders. This study aims to investigate the molecular mechanism of miR-128 in STC. Methods: A murine model of STC was constructed based on morphine and rhubarb. MiRNA array, bioinformatic analysis and experimental validation were applied to confirm the miR-128 and its target signaling pathway. The expression of miR-128 and downstream genes analysis were performed in the models. Inflammatory cytokine release in response to miR-128 regulation was evaluated in vitro. Findings: The expression of stem cell factor (SCF) and neuron-specific enolase (NSE) were low in the STC model. In addition, STC group showed a significant downregulation of miR-128 and upregulation of p38α and macrophage colony stimulating factors (M-CSF). Moreover, we observed elevated inflammatory cytokine and decreased anti-inflammatory cytokine levels in colonic tissues. Furthermore, co-culture assays indicated that regulating expression of miR-128 in colonic epithelial cells induced the secretion of IL-6 and TNF-α by macrophages. Interpretation: Our study demonstrated that miR-128 regulated the p38α/M-CSF signaling pathway to promote chronic inflammatory responses and changes in the immune microenvironment of the colon, thereby offering potential insights into the pathogenesis of STC and therapeutic targets for its treatment. Funding Information: This work was supported by grants from the National Natural Science Foundation of China (No.81570492, No.81500505), Science and Technology Innovation Fostering Foundation of Zhongnan Hospital, Wuhan University (cxpy2017018), Medical Science and Technology Innovation Platform of Health Commission of Hubei Province /Zhongnan Hospital of Wuhan University (No.PTXM2019011), Health Commission of Hubei Province (No. WJ2019M206), Science and Technology Department of Hubei Province (No.2018CKB913), and the Clinical Research Special Fund of Wu Jieping Medical Foundation (No.320.6750.18467, No.320.6750.19089-14). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: Experiments were performed under a project license (No.2018039) granted by the ethics committee of Zhongnan Hospital of Wuhan University, Wuhan, China, in compliance with Animal Research Center of Wuhan University guidelines for the care and use of animals.

Sustained release of PKR inhibitor C16 from mesoporous silica nanoparticles significantly enhances mRNA translation and anti-tumor vaccination

In this study, we developed a mesoporous silica nanoparticles - mRNA (MSN-mRNA) subcutaneous delivery system composed of naked mRNA and a subcutaneous depot of imidazolo-oxindole RNA-activated protein kinase (PKR) inhibitor C16. We show that C16 treatment during mRNA transfection is a potent immune evasion approach that non-linearly enhances translation of unmodified mRNA in both mouse fibroblasts and dendritic cells in vitro exceeding that of nucleoside-modified mRNA. Notably, C16 further enhances translation of nucleoside-modified mRNA and HPLC purified mRNA. However, translation enhancement is dependent on and potentiated by C16′s continuous presence. C16 mediated translation enhancement is extended in vivo by employing MSN as an interface to sustain-release C16. Subcutaneously administered MSN-mRNA significantly enhanced in vivo translation and expression kinetics of naked mRNA in unmodified, nucleoside-modified, and HPLC purified formats. We applied a MSN-mRNA vaccine formulation composed of naked mRNA encoding ovalbumin and granulocyte macrophage colony stimulating factor, and C16@MSNs on a xenograft E.G7-OVA prophylactic tumor model, resulting in very potent tumor inhibition. The MSN-mRNA delivery system bears great translational potential in mRNA therapeutics.

In Vitro Evaluation of [3H]CPPC as a Tool Radioligand for CSF-1R.

Microglia play a role in several central nervous system (CNS) diseases and are a highly sought target for positron emission tomography (PET) imaging and therapeutic intervention. 5-Cyano-N-(4-(4-[11C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide ([11C]CPPC) is a radiopharmaceutical designed to selectively target microglia via macrophage colony stimulating factor-1 receptor (CSF-1R) in the CNS. Herein, we report the first preclinical evaluation of [3H]CPPC using radioligand binding methods for the evaluation of putative CSF-1R inhibitors in rodent models of neuroinflammation. The distribution of [3H]CPPC by autoradiography did not align with 18 kDa translocator protein (TSPO) distribution using [3H]PBR28 and IBA-1 staining for microglia. In the CNS, [3H]CPPC had considerable nonspecific binding, as indicated by a low displacement of the tritiated ligand by unlabeled CPPC and the known CSF1R inhibitors BLZ-945 and PLX3397. Spleen was identified as a tissue that provided an adequate signal-to-noise ratio to enable screening with [3H]CPPC and a library of 20 novel PLX3397 derivatives. However, unlabeled CPPC lacked selectivity and showed off-target binding to a substantial number of kinase targets (204 out of 403 tested) at a concentration relevant to in vitro radioligand binding assays (10 μM). These findings suggest that, while [3H]CPPC may have utility as a radioligand tool for the evaluation of peripheral targets and screening of CSF-1R inhibitors, it may have limited utility as an in vivo CNS imaging probe on the basis of the current evaluation.

Influence of administration of mesenchymal stromal cell on pediatric oxygenator performance and inflammatory response

ObjectiveMesenchymal stromal cells have important immunomodulatory and neuroprotective properties. The aim of this study was to evaluate the feasibility of mesenchymal stromal cell administration into a cardiopulmonary bypass (CPB) circuit, including a pediatric oxygenator, and to assess the immunomodulatory response of the circulating blood prime. MethodsA bypass circuit with a pediatric oxygenator, including integral filter was primed with bank whole blood. Normal saline (control) or 120 × 106 mesenchymal stromal cells were injected into the venous reservoir after 80 minutes of perfusion. To assess oxygenator function, immune reaction, and cytokine/chemokine levels, the ex vivo circulation was maintained for 300 minutes after administration. ResultsThere were no differences in flow rate, trans-oxygenator pressure gradient, blood oxygen, and carbon dioxide levels between control and cell delivery groups. No adhesion of mesenchymal stromal cells was observed on the filter mesh by scanning electron microscopy. Lymphocyte surface marker assay found no difference in the number of B cells, T cells, or natural killer cells between the 2 groups, indicating no immunogenicity of allogeneic mesenchymal stromal cells under ex vivo CPB conditions. CPB significantly changed the level of interleukin (IL) 4, IL-6, IL-8, IP-10, macrophage colony stimulating factor, macrophage inflammatory protein-1β, monocyte chemoattractant protein-1, and IL-1α over time. IL-6 level was significantly increased after cell administration. ConclusionsThe administration of mesenchymal stromal cells does not interfere with oxygenator function. Allogeneic mesenchymal stromal cells show no immunogenicity, and increase plasma IL-6 level during ex vivo circulation. Further investigation is necessary to determine the effect of mesenchymal stromal cell delivery through CPB during pediatric cardiac surgery.

Stabilization of C/EBPβ through direct interaction with STAT3 in H-Ras transformed human mammary epithelial cells

Signal transducer and activator of transcription 3 (STAT3) plays important roles in cancer-associated inflammation by controlling expression of proinflammatory cytokines and chemokines. Recent studies suggest that C/EBPβ (CCAAT-enhancer binding protein beta) and STAT3 synergistically stimulate cancer cell proliferation and epithelial-mesenchymal transition. C/EBPβ is a leucine-zipper transcription factor that regulates expression of a variety of inflammatory cytokines or chemokines, such as IL-8, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor) which induce neutrophil infiltration and differentiation. However, molecular mechanisms by which STAT3 and C/EBPβ cooperatively interact had not been fully elucidated. In this study, we found that the level of C/EBPβ protein, but not that of its mRNA transcript, was decreased in the absence of STAT3 in H-Ras transformed human mammary epithelial (H-Ras MCF10A) cells. In addition, silencing STAT3 dramatically induced ubiquitination of C/EBPβ for proteasomal degradation. Furthermore, direct interaction between STAT3 and C/EBPβ was confirmed by immunoprecipitation and proximity ligation assays. Taken together, these results suggest that STAT3 stabilizes C/EBPβ, thereby promoting cancer-associated inflammation.

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1

Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.

Transcriptomic Analysis of Rat Macrophages.

The laboratory rat is widely used as a model for human diseases. Many of these diseases involve monocytes and tissue macrophages in different states of activation. Whilst methods for in vitro differentiation of mouse macrophages from embryonic stem cells (ESC) and bone marrow (BM) are well established, these are lacking for the rat. The gene expression profiles of rat macrophages have also not been characterised to the same extent as mouse. We have established the methodology for production of rat ESC-derived macrophages and compared their gene expression profiles to macrophages obtained from the lung and peritoneal cavity and those differentiated from BM and blood monocytes. We determined the gene signature of Kupffer cells in the liver using rats deficient in macrophage colony stimulating factor receptor (CSF1R). We also examined the response of BM-derived macrophages to lipopolysaccharide (LPS). The results indicate that many, but not all, tissue-specific adaptations observed in mice are conserved in the rat. Importantly, we show that unlike mice, rat macrophages express the CSF1R ligand, colony stimulating factor 1 (CSF1).

Macrophage colony-stimulating factor could evaluate both disease activity and renal involvement in systemic lupus erythematosus.

Aberrations of monocyte/macrophage-associated cytokines are increasingly recognized in systemic lupus erythematosus (SLE). However, the combined expression of these cytokines has not been sufficiently studied in relation to either disease activity or renal involvement in SLE. Here, we explored clinical values of monocyte/macrophage-associated cytokines for monitoring of disease activity and renal involvement in SLE patients. A total of 44 healthy people and 100 SLE patients were enrolled in this study. The serum levels of 8 monocyte/macrophage-associated cytokines [interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), macrophage colony stimulating factor (M-CSF), interleukin-34 (IL-34), interleukin-10 (IL-10), and urokinase-type plasminogen activator receptor (uPAR)] were measured, and correlations between their levels and both SLE activity and renal involvement were analyzed using receiver operating characteristic (ROC) curves. Additionally, we analyzed the correlation between M-CSF level and laboratory or clinical data and used real time-polymerase chain reaction (RT-PCR) to assess M-CSF messenger RNA (mRNA) expression in sorted candidate cells. The levels of IL-6, IFN-γ, and TNF-α were significant for predicting SLE activity, while the M-CSF level was significant for predicting both SLE activity and renal involvement. Furthermore, the mRNA expression of M-CSF in monocytes from SLE patients was higher than that of healthy people, and the M-CSF level was positively correlated with monocyte numbers. The cytokine M-CSF is a promising marker to evaluate both disease activity and renal involvement in SLE, and the high level of M-CSF in SLE patients was mainly derived from monocytes.

Negative regulation of FOXP3 expression by c-Rel O-GlcNAcylation.

O-GlcNAcylation is a reversible post-translational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, nuclear factor kappaB c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2, interferon gamma and granulocyte macrophage colony stimulating factor in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3)-the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin-induced diabetes and spontaneous diabetes in nonobese diabetic mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.

High-density lipoprotein modulates tumor-associated macrophage for chemoimmunotherapy of hepatocellular carcinoma

Intratumoral abundance of alternatively activated macrophages (M2) in hepatocellular carcinoma (HCC) is associated with advanced stages, poor prognosis and failure of checkpoint blockade immunotherapy. In this work, we discover that synthetic high-density lipoproteins (sHDLs) preferentially deliver cargoes into M2 and Hepa1–6 HCC cells, based on which finding we create a functional sHDL containing esterase-responsive prodrugs of vadimezan and gemcitabine (VG-sHDLs). The VG-sHDLs induce the HCC cells to release damage-associated molecular patterns (high mobility group box 1 protein and ATP) and cytokines including macrophage colony stimulating factor and type I interferons, improving monocyte differentiation into classically activated macrophages (M1) and dendritic cells. Meanwhile, the VG-sHDLs kill M2 without significant toxicity to M1. After intravenous injection, the VG-sHDLs increase intratumoral M1 as well as proinflammatory molecules such as C-X-C motif ligand 9 and 10 and interleukin 12, but reduce M2 and anti-inflammatory cytokine interleukin 10. These changes enhance the recruitment and activity of cytotoxic T lymphocytes, which eradicate HCC tumors and also establish tumor-specific immune memory that prevents recurrence. This work sheds light on the interactions between sHDL and intratumoral cells, and provides rationale for chemoimmunotherapy of HCC using conventional chemotherapeutics and emerging immune modulators.

A decrease in PPD specific CD4 T cell CD38 and HLA-DR expression in pulmonary tuberculosis patients after 8 weeks of therapy correlates with successful anti-tuberculosis treatment

Tuberculosis (TB) is a major health problem in Indonesia with a million new cases each year. The CD4 T cell adaptive immune response against Mycobacterium tuberculosis (MTB) is central to the control of this disease. We investigated whether standard therapy of TB causes changes to these cells in the early stages of treatment. To do this we took blood samples from 2 groups of TB patients in Banda Aceh, Indonesia; one from a group of patients before treatment, and the other from a group who become smear negative after 8 weeks treatment. MTB specific CD4 T cells were identified by ex vivo stimulation with PPD and flow cytometric measurement of intracellular cytokines and surface markers. We found no difference in total PPD specific CD4 T cells between the groups, but that the proportion of these cells CD38 + HLA-DR+ was significantly lower in the treatment group. This decrease was not specific to Interferon gamma (IFNg), Interleukin-2 (IL-2) or Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) producing cells. Our findings show that anti-MTB treatment affects the adaptive immune response, and that measuring the decrease of the PPD specific CD4 T cell CD38+HLA-DR+ phenotype could be a useful parameter for determination of treatment success.

Mechanotransduction via a TRPV4-Rac1 signaling axis plays a role in multinucleated giant cell formation

Multinucleated giant cells are formed by the fusion of macrophages and are a characteristic feature in numerous pathophysiological conditions including the foreign body response (FBR). Foreign body giant cells (FBGCs) are inflammatory and destructive multinucleated macrophages and may cause damage and/or rejection of implants. However, while these features of FBGCs are well established, the molecular mechanisms underlying their formation remain elusive. Improved understanding of the molecular mechanisms underlying the formation of FBGCs may permit the development of novel implants that eliminate or reduce the FBR. Our previous study showed that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel/receptor, is required for FBGC formation and FBR to biomaterials. Here, we have determined that (a) TRPV4 is directly involved in fusogenic cytokine (interleukin-4 plus granulocyte macrophage–colony stimulating factor)–induced activation of Rac1, in bone marrow–derived macrophages; (b) TRPV4 directly interacts with Rac1, and their interaction is further augmented in the presence of fusogenic cytokines; (c) TRPV4-dependent activation of Rac1 is essential for the augmentation of intracellular stiffness and regulation of cytoskeletal remodeling; and (d) TRPV4-Rac1 signaling axis is critical in fusogenic cytokine–induced FBGC formation. Together, these data suggest a novel mechanism whereby a functional interaction between TRPV4 and Rac1 leads to cytoskeletal remodeling and intracellular stiffness generation to modulate FBGC formation.

Anaplazmozisli Koyunlarda Bazı Inflamasyon Markırlarının Araştırılması

Anaplazmozis koyunlarda siklikla gorulen, kan hucrelerini enfekte eden artropod kaynakli ve ekonomik kayiplara neden olabilen paraziter bir hastaliktir. Bu calisma Anaplazmozis ile enfekte koyunlarda bazi serum inflamasyon markir seviyelerini degerlendirmek icin planlanmistir. Yapilan arastirma icin kullanilacak olan kan ornekleri, Van Buyuksehir Belediyesi Mezbahanesi’ne getirilen 20 saglikli ve 20 Anaplazmozis’li koyundan saglandi. Kontrol ve hasta gruplarinin belirlenmesi, hastaligin klinik belirtileri, giemsa boyali kan frotileri ve serolojik yontem (cELISA) yardimiyla yapildi. Toplanan 91 koyunlarin ait serum ornegi Anaplazma spp. turlerinin antikorlarinin varligi yonunde serolojik olarak incelendiginde %73.6’si (67/91) Anaplazmozis yonunden seropozitif bulundu. Kan frotisinde Anaplazma etkenleri gorunen ve ELISA inhibisyon degeri yuksek cikan koyunlarin kanlari analizler icin kullanildi. Anaplazmozis’li koyunlar teshis edildikten sonra pozitif ve negatif olarak gruplandirildi. Pozitifligi tespit edilen 20 koyun serumunda granulosit makrofaj koloni uyarici faktor (GM-CSF), interloykin-1β (IL-1β), interloykin-6 (IL-6), tumor nekroz faktor- alfa (TNF-α) duzeyleri elisa kiti ile kit prosedurune gore belirlendi. Proinflamasyon parametrelerinden serum IL-1β (ng/L), TNF-α (ng/L), GM-CSF (ng/L) duzeylerinin negatif gruba gore istatiksel olarak yukseldigi tespit edildi. Anaplazmaya karsi gelisen inflamasyon ile ilgili mekanizmalarin incelenmesi, ileride gelistirilecek immunoterapi arastirmalari icin Anaplazma enfeksiyonlarinin teshis ve onlemeye yonelik calismalara katki saglayacagi dusunulmektedir.

Bifunctional GM-CSF-derived peptides as tools for O-glycoengineering and protein tagging

Rapid development of effective biotherapeutics has been a concern during the last couple decades. In our work we designed two novel peptide tags, GMOP and mGMOP, derived from the N-terminal region of human granulocyte and macrophage colony stimulating factor (hGM-CSF), which contain four and six potential O-glycosylation sites, respectively. These peptide tags were fused to the N-terminus of human interferon-α2b (hIFN-α2b), a therapeutic antiviral and antiproliferative protein rapidly cleared from circulation. Two new molecules were obtained which, consistently with the presence of O-glycans, showed higher molecular masses, more negatively charged isoforms, and higher sialic acid content compared to wild-type IFN. In vitro bioactivity of purified chimeras revealed a similar antiviral specific biological activity (SBA) compared to unmodified IFN. A reduction of antiproliferative SBA was only observed for mGMOP-IFN. Pharmacokinetic studies in rats showed a notable improvement in terminal half-life (t1/2elim) (3.3 and 2.8 times-longer) and a marked reduction of the apparent clearance (CLapp, 3.7 and 4.1-fold lower for GMOP-IFN and mGMOP-IFN in comparison with native IFN, respectively). Furthermore, the in vitro thermal and plasma stability of both proteins was improved. Finally, a monoclonal antibody (mAb) that recognizes an N-terminal GM-CSF epitope was able to bind both chimeras in western blots and ELISAs. This demonstrates the potential of both peptides to behave as bifunctional tags to create novel long-acting biotherapeutics and to facilitate detection and purification.

The inhibitory effect of melatonin on osteoclastogenesis of RAW 264.7 cells in low concentrations of RANKL and MCSF.

RAW 264.7 cells are one of the most recommended cell lines for investigating the activity and differentiation of osteoclasts. These cells differentiate into osteoclasts in the presence of two critical components: receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (MCSF). Melatonin (MEL) hormone has recently become one of the small molecules used in the field of bone regeneration and bone disease treatment, as it has the ability to inhibit the differentiation of osteoclasts directly by suppression of the NF-κB signaling pathway. The main aim of the current study is to determine sufficient RANKL/MCSF concentrations for differentiation of the cells to osteoclasts and to describe the repressive effect of MEL on the osteoclastogenesis of these cells. In this regard, it was found that 10 ng/mL of RANKL- and MCSF-containing medium is suitable for inducing osteoclastogenesis of the cells. In addition, melatonin at doses in the range of 100–1000 µM does not have a cytotoxic effect. Subsequently, results of tartrate resistant acid phosphatase (TRAP) activity, TRAP staining, and relative expressions of cathepsin K, nuclear factor of activated T cells one (NFATC1), and TRAP genes showed a suppressive effect of MEL —especially 800 µM— on RANKL-induced osteoclastogenesis of these cells.