Rat Bone Marrow-derived Macrophages Research Articles

Differential effects of macrophage subtype-specific cytokines on fibroblast proliferation and endothelial cell function in co-culture system.

Macrophages are involved in several critical activities associated with tissue repair and regeneration. Current approaches in regenerative medicine are focusing on leveraging the innate immune response to accelerate tissue regeneration and improve long-term healing outcomes. Of particular interest in this regard are the currently known, four main M2 macrophage subtypes: M2interleukin (IL)-4,IL-13, M2IC, M2IL-10, M2non-selective adenosine receptor agonists (NECA) (M2IL-4,IL-13 → M2NECA). In this study, rat bone marrow-derived macrophages (M0) were polarized to each of the four subtypes M2IL-4,IL-13 → M2NECA and cultured for 72 h in vitro. Luminex assay results highlighted increased production of tissue inhibitor of metalloproteinases-1 (TIMP-1) for M2IL-4,IL-13, higher amounts of transforming growth factor-beta 1 (TGF-β1) for M2IL-10, and elevated vascular endothelial growth factor A (VEGF-A) from M2NECA. Co-culture experiments performed with M2IL-10 macrophages and L929 fibroblasts highlighted the increased production of soluble collagen within the media as well as higher amounts of collagen in the extracellular matrix. Human umbilical vein endothelial cells (HUVECs) were co-cultured with M2NECA macrophages, which demonstrated an increase in intercellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM), as well as increased formation of endothelial tubes. The findings of this study emphasize a critical demand for further characterization and analyses of distinct M2 subtypes and careful selection of specific macrophage populations for regeneration of specific tissue types. The current, broad classification of "M2" may be sufficient in many general tissue engineering applications, but, as conditions are constantly in flux within the microenvironment in vivo, a higher degree of specificity and control over the initial M2 subtype could result in more consistent long-term outcomes where macrophages are utilized as part of an overall regenerative strategy.

Adipose-derived stem cells transplantation improves survival and alleviates contraction of skin grafts via promoting macrophages M2 polarization.

Full-thickness skin grafts are widely used in plastic and reconstructive surgery. The main limitation of skin grafting is the poor textural durability and associated contracture, which often needs further corrective surgery. Excessive inflammation is the main reason for skin graft contractions, which involve overactivation of myofibroblasts. These problems have prompted the development of new therapeutic approaches, including macrophage polarization modulation and stem cell-based therapies. Currently, adipose-derived stem cells (ASCs) have shown promise in promoting skin grafts survival and regulating macrophage phenotypes. However, the roles of ASCs on macrophages in decreasing skin grafts contraction remain unknown. Rat adipose-derived stem cells (rASCs) were isolated from rat inguinal adipose tissues. Full-thickness skin graft model was constructed on male rats divided into control group and rASCs treatment group. Skin graft was assessed for concentration, elasticity modulus and stiffness. Rat bone marrow-derived macrophages (rBMDMs) were isolated from rat femurs, and subsequent RT-qPCR and coculture assays were carried out to explore the cellular mechanisms. Immunohistochemical and immunofluorescence staining were used to verify mechanisms in vivo. In vivo results showed that after injection of ASCs, improved texture, increased survival and inhibited contraction of skin grafts were seen. Vascularization was also improved as illustrated by laser perfusion image and vascular endothelial growth factor (VEGF) concentration. Histological analysis revealed that ASCs injection significantly reduced expression of pro-inflammatory cytokines (TNF-a, IL-1β) and increased expression of anti-inflammatory (IL-10) and pro-healing cytokines (IGF-1). At cellular level, after co-culturing with rASCs, rat bone marrow derived macrophages (rBMDMs) favored M2 polarization even under inflammatory stimulus. ASCs treatment enhanced vascularization via angiogenic cytokines secretion and alleviated inflammatory environment in skin grafts by driving M2 macrophages polarization, which improved survival and decreased skin grafts contraction. Our work showed that ASCs transplantation can be harnessed to enhance therapeutic efficacy of skin grafting in cutaneous defects treatment.

CGRP promotes osteogenic differentiation by regulating macrophage M2 polarization through HDAC6/AKAP12 signaling pathway.

Aim: To determine the mechanism of Calcitonin gene-related peptide (CGRP) in bone healing.Materials & methods: Alkaline phosphatase (ALP) activity and inflammatory-factor levels were detected usingELISA. Osteogenic differentiation was assessed using Alizarin red staining technique. The interaction between histone deacetylase 6 (HDAC6) and A-kinase anchoring protein 12 (AKAP12) was investigated through Co- immunoprecipitation.Results: CGRP treatment promoted rat bone marrow-derived macrophages (BMDMs) M2 polarization. CGRP facilitated osteogenic differentiation by enhancing M2 polarization of BMDMs. Mechanistically, CGRP promoted AKAP12 acetylation to activate the extracellular regulated protein kinases pathway by HDAC6 inhibition.Conclusion: CGRP promoted M2 polarization of rat BMDMs and facilitated osteogenic differentiation through the HDAC6/AKAP12/extracellular regulated protein kinases signaling pathway, thereby promoting bone healing.

Ginsenoside Rg1 reduces cardiac inflammation against myocardial ischemia/reperfusion injury by inhibiting macrophage polarization

BackgroundMyocardial ischemia/reperfusion (MI/R) injury is the main cause of death worldwide and poses a significant threat to cardiac health. Ginsenoside Rg1 has been shown to have inhibitory effects on inflammatory activation, oxidative stress, and cardiac injury, suggesting that Rg1 may have therapeutic effects on MI/R injury. However, the mechanism remains to be further studied. Materials and methodsLeft anterior descending coronary artery ligation was performed in Sprague-Dawley rats to construct an MI/R model in vivo. Organ index, electrocardiogram, infarct size, histopathological changes, and detection of cardiac injury and inflammatory factors in the rats were used to evaluate myocarditis, macrophage polarization, and fibrosis. We also used rat bone marrow-derived macrophages (BMDMs) to further investigate the effects of Rg1 on absent in melanoma 2 (AIM2) activation and macrophage polarization in vitro. ResultsAdministration of Rg1 exhibited dose-dependent cardioprotective effects and effectively reduced MI/R injury. Rg1 significantly attenuated myocardial inflammation and inhibited M1 macrophage polarization during MI/R injury. Furthermore, Rg1 significantly reduced cardiac fibrosis in response to MI/R injury. This anti-fibrotic effect may contribute to the preservation of cardiac structure and function following an ischemic insult. Meanwhile, Rg1 effectively inhibited the activation of the AIM2 inflammasome in vitro, highlighting its potential as a key regulator of inflammatory pathways. ConclusionOur findings elucidate the multifaceted mechanisms underlying Rg1's cardioprotective effects, including its ability to mitigate inflammation, modulate macrophage polarization, and inhibit fibrosis.

Quercetin as a promising intervention for rat osteoarthritis by decreasing M1-polarized macrophages via blocking the TRPV1-mediated P2X7/NLRP3 signaling pathway.

Osteoarthritis (OA) is characterized by an imbalance between M1 and M2 polarized synovial macrophages. Quercetin has shown protective effects against OA by altering M1/M2-polarized macrophages, but the underlying mechanisms remain unclear. In this study, rat chondrocytes were treated with 10 ng/mL of IL-1β. To create M1-polarized macrophages in vitro, rat bone marrow-derived macrophages (rBMDMs) were treated with 100 ng/mL LPS. To mimic OA conditions observed in vivo, a co-culture system of chondrocytes and macrophages was established. ATP release assays, immunofluorescence assays, Fluo-4 AM staining, Transwell assays, ELISA assays, and flow cytometry were performed. Male adult Sprague-Dawley (SD) rats were used to create an OA model. Histological analyses, including H&E, and safranin O-fast green staining were performed. Our data showed a quercetin-mediated suppression of calcium ion influx and ATP release, with concurrent downregulation of TRPV1 and P2X7 in the chondrocytes treated with IL-1β. Activation of TRPV1 abolished the quercetin-mediated effects on calcium ion influx and ATP release in chondrocytes treated with IL-1β. In the co-culture system, overexpression of P2X7 in macrophages attenuated the quercetin-mediated effects on M1 polarization, migration, and inflammation. Either P2X7 or NLRP3 knockdown attenuated IL-1β-induced M1/M2 polarization, migration, and inflammation. Moreover, overexpression of TRPV1 reduced the quercetin-mediated suppressive effects on OA by promoting M1/M2-polarized macrophages in vivo. Collectively, our data showed that quercetin-induced suppression of TRPV1 leads to a delay in OA progression by shifting the macrophage polarization from M1 to M2 subtypes via modulation of the P2X7/NLRP3 pathway.

MiR-345-3p Modulates M1/M2 Macrophage Polarization to Inhibit Inflammation in Bone Infection via Targeting MAP3K1 and NF-κB Pathway.

Infection after fracture fixation (IAFF), a complex infectious disease, causes inflammatory destruction of bone tissue and poses a significant clinical challenge. miR-345-3p is a biomarker for tibial infected nonunion; however, the comprehensive mechanistic role of miR-345-3p in IAFF is elusive. In this study, we investigated the role of miR-345-3p in IAFF pathogenesis through invivo and invitro experiments. In vivo, in a rat model of IAFF, miR-345-3p expression was downregulated, accompanied by increased M1 macrophage infiltration and secretion of proinflammatory factors. In vitro, LPS induced differentiation of primary rat bone marrow-derived macrophages into M1 macrophages, which was attenuated by miR-345-3p mimics. miR-345-3p promoted M1 to M2 macrophage transition-it reduced the expression of cluster of differentiation (CD) 86, inducible NO synthase, IL-1β, and TNF-α but elevated those of CD163, arginase-1, IL-4, and IL-10. MAPK kinase kinase 1 (MAP3K1), a target mRNA of miR-345-3p, was overexpressed in the bone tissue of IAFF rats compared with that in those of the control rats. The M1 to M2 polarization inhibited MAP3K1 signaling pathways invitro. Conversely, MAP3K1 overexpression promoted the transition from M2 to M1. miR-345-3p significantly inhibited NF-κB translocation from the cytosol to the nucleus in a MAP3K1-dependent manner. In conclusion, miR-345-3p promotes the polarization of M1 macrophages to the M2 phenotype by inhibiting the MAP3K1 and NF-κB pathways. These findings provide insight into the pathogenesis and immunotherapeutic strategies for IAFF and offer potential new targets for subsequent research.

10-Residue MyD88-Peptide Adopts β-Sheet Structure, Self-Assembles, Binds to Lipopolysaccharides, and Rescues Mice from Endotoxin-Mediated Lung-Infection and Death.

Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel β-sheet AMPs, we identified two short cationic amphipathic β-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine-valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with β-sheet structures and self-assembling properties.

PD-1/PD-L1 inhibition promotes hepatic regeneration in small-for-size liver following extended hepatectomy.

Small-for-size syndrome following liver surgery is characterized by compromised liver regeneration. Liver macrophages play key roles in initiating liver regeneration, and modulation of the immune microenvironment through macrophages may accelerate liver regeneration. In our current study, we aimed to explore the involvement of innate immunity after extended hepatectomy in rats and humans, and to test the effect of immunity modulation on small-for-size liver regeneration in rats. Serum programmed cell death protein ligand 1 (PD-L1) was measured after major hepatectomy and minor hepatectomy in humans and rats. Liver regeneration in rats was assessed using liver-to-body weight ratio and kinetic growth rate, antigen Ki67 and proliferating cell nuclear antigen (PCNA), and macrophage polarization was assessed by inducible nitric oxide synthase (iNOS), cluster of differentiation protein 163 (CD163) expression by immunohistochemistry (IHC) and iNOS/CD163 ratio. Rat hepatocyte BRL or human hepatocyte LO2 were co-cultured with rat bone marrow-derived macrophages or human macrophages THP-1. BMS-1 or Nivolumab were used to block programmed cell death protein 1 (PD-1)/PD-L1 in vitro and in vivo. PD-L1 expressions were significantly higher following major hepatectomy compared to minor resection in both humans and rats; compromised liver regeneration after extended hepatectomy in rats was associated with PD-L1 upregulation and M2 macrophage polarization. M1 macrophages increased proliferation of hepatocytes through interleukin-6 (IL-6), and M2 macrophages decreased hepatocyte proliferation; blocking PD-1/PD-L1 reversed the effect of M2 macrophages on the survival of hepatocytes in vitro and promoted liver growth in rats through M1 macrophage polarization. Compromised hepatic regeneration following extended hepatectomy is characterized by M2 macrophage polarization and upregulated PD-L1 expression. Blocking PD-1/PD-L1 may enhance small-for-size liver regeneration by inducing M1 macrophage polarization.

Application of Bone Marrow-Derived Macrophages Combined with Bone Mesenchymal Stem Cells in Dual-Channel Three-Dimensional Bioprinting Scaffolds for Early Immune Regulation and Osteogenic Induction in Rat Calvarial Defects.

The host immune response to biomaterials is critical for determining scaffold fate and bone regeneration outcomes. Three-dimensional (3D) bioprinted scaffolds encapsulated with living cells can improve the inflammatory microenvironment and further accelerate bone repair. Here, we screened and adopted 8% methacrylamidated gelatin (GelMA)/1% methacrylamidated hyaluronic acid (HAMA) as the encapsulation system for rat bone marrow-derived macrophages (BMMs) and 3% Alginate/0.5 mg/mL graphene oxide (GO) as the encapsulation system for rat bone mesenchymal stem cells (BMSCs), thus forming a dual-channel bioprinting scaffold. The 8% GelMA/1% HAMA/3% Alginate/0.5 mg/mL GO (8/1/3/0.5) group could form a scaffold with a stable structure, good mechanical properties, and satisfied biocompatibility. When exploring the crosstalk between BMMs and BMSCs in vitro, we found that BMSCs could promote the polarization of BMMs to M2 type at the early stage, reduce the pro-inflammatory gene expression, and increase anti-inflammatory gene expression; conversely, BMMs can promote the osteogenic differentiation of BMSCs. In addition, in the model of rat calvarial defects, the dual-channel scaffold encapsulated with BMMs and BMSCs was more effective than the single-cell scaffold and the acellular scaffold. The paracrine of BMMs and BMSCs in the biodegradable dual-channel scaffold effectively promoted the M2-type polarization of macrophages in the microenvironment of early bone defects, avoided excessive inflammatory responses, and further promoted bone repair. In conclusion, our findings suggested that using 3D bioprinting to simultaneously encapsulate two primary cells of BMMs and BMSCs in a dual-channel system may be an effective way to promote bone repair from the perspective of early immune regulation and late induction of osteogenesis.

Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel.

Transient receptor potential vanilloid 2 (TRPV2) is a multimodal ion channel implicated in diverse physiopathological processes. Its important involvement in immune responses has been suggested such as in the macrophages' phagocytosis process. However, the endogenous signaling cascades controlling the gating of TRPV2 remain to be understood. Here, we report that enhancing tyrosine phosphorylation remarkably alters the chemical and thermal sensitivities of TRPV2 endogenously expressed in rat bone marrow-derived macrophages and dorsal root ganglia (DRG) neurons. We identify that the protein tyrosine kinase JAK1 mediates TRPV2 phosphorylation at the molecular sites Tyr(335), Tyr(471), and Tyr(525). JAK1 phosphorylation is required for maintaining TRPV2 activity and the phagocytic ability of macrophages. We further show that TRPV2 phosphorylation is dynamically balanced by protein tyrosine phosphatase non-receptor type 1 (PTPN1). PTPN1 inhibition increases TRPV2 phosphorylation, further reducing the activation temperature threshold. Our data thus unveil an intrinsic mechanism where the phosphorylation/dephosphorylation dynamic balance sets the basal chemical and thermal sensitivity of TRPV2. Targeting this pathway will aid therapeutic interventions in physiopathological contexts.

Polyetheretherketone fibers woven fabrics coated nanostructured silicon nitride and load EGCG as artificial ligaments for promoting ligament-bone integration

Design of an artificial ligament with osteogenic activity to promote ligament-bone integration for anterior cruciate ligament (ACL) reconstruction remains a huge challenge. In this work, polyetheretherketone fibers with nanostructured silicon nitride coating (PSN) and loading epigallocatechin gallate (EGCG) were woven into fabrics (PSN@EG) for artificial ligament application.The in vitro experiments confirmed that PSN@EG significantly upregulated M2 macrophage polarization that promoted secretory of anti-inflammatory cytokines while downregulated M1 macrophage polarization that inhibithed secretory of pro-inflammatory cytokines. Moreover, PSN@EG remarkably improved osteoblastic differentiation of rat bone marrow mesenchymal stem cells (BMSC) while suppressed osteoclastic differentiation of rat bone marrow-derived macrophages (BMM) in macrophage condition medium. The in vivo experiments revealed that PSN@EG promoted new bone formation for ligament-bone integration that inhibited fibrous encapsulation formation. It could be suggested that the synergistic effects of the optimized surface charactieristics (e.g., nano-micro topography), and sustained release of Si ions and EGCG caused the immunomodulatory of PSN@EG, which improved pro-osteogenic macrophage polarization that provided a favorable anti-inflammatory immune environment for enhancing osteogenesis while inhibiting osteoclastogenesis. In summary, PSN@EG with immunomodulatory significantly promoted osteoblastic differentiation while suppressed osteoclastic differentiation, thereby facilitating osteogenesis for ligament-bone integration. As a novel artificial ligament, PSN@EG has tremendous potential for ACL reconstruction.

Interleukin-20 Acts as a Promotor of Osteoclastogenesis and Orthodontic Tooth Movement

Objectives Bones constitute organs that are engaged in constant self-remodelling. Osteoblast and osteoclast homeostasis during remodelling contribute to overall skeletal status. Orthodontics is a clinical discipline that involves the investigation and implementation of moving teeth through the bone. The application of mechanical force to the teeth causes an imbalance between osteogenesis and osteogenesis in alveolar bone, leading to tooth movement. Osteoimmunology comprises the crosstalk between the immune and skeletal systems that regulate osteoclast–osteoblast homeostasis. Interleukin- (IL-) 20, an IL-10 family member, is regarded as a proinflammatory factor for autoimmune diseases and has been implicated in bone loss disease. However, the mechanism by which IL-20 regulates osteoclast differentiation and osteoclastogenesis activation remains unclear. This study investigated the effects of IL-20 on osteoclast differentiation in a rat model; it explored the underlying molecular mechanism in vitro and the specific effects on orthodontic tooth movement in vivo. Methods For in vitro analyses, primary rat bone marrow-derived macrophages (BMMs) were prepared from Sprague–Dawley rats for osteoclast induction. After BMMs had been treated with combinations of recombinant IL-20 protein, siRNA, and plasmids, the expression levels of osteoclast-specific factors and signalling pathway proteins were detected through real-time polymerase chain reaction, western blotting, and immunofluorescence staining. For in vivo analyses, IL-20 was injected into the rat intraperitoneal cavity after the establishment of a rat orthodontic tooth movement (OTM) model. OTM distance was detected by Micro-CT and HE staining; the expression levels of protein were detected through immunofluorescence staining. Results In vitro analyses showed that a low concentration of IL-20 promoted preosteoclast proliferation and osteoclastogenesis. However, a high concentration of IL-20 inhibited BMM proliferation and osteoclastogenesis. IL-20 knockdown decreased the expression of osteoclast specific-markers, while IL-20 overexpression increased the expression of osteoclast specific-markers. Furthermore, IL-20 regulated osteoclast differentiation through the OPG/RANKL/RANK pathway. Overexpression of IL-20 could significantly upregulate RANKL-mediated osteoclast differentiation and osteoclast specific-marker expression; moreover, RANKL/NF-κB/NFATc1 acted as downstream signalling molecule for IL-20. In vivo analysis showed that OTM speed was significantly increased after intraperitoneal injection of IL-20; additionally, mechanical stress sensing proteins were markedly activated. Conclusions IL-20 augments osteoclastogenesis and osteoclast-mediated bone erosion through the RANKL/NF-κB/NFATc1 signalling pathway. IL-20 inhibition can effectively reduce osteoclast differentiation and diminish bone resorption. Furthermore, IL-20 can accelerate orthodontic tooth movement and activate mechanical stress sensing proteins.

Bi-directional regulation functions of lanthanum-substituted layered double hydroxide nanohybrid scaffolds via activating osteogenesis and inhibiting osteoclastogenesis for osteoporotic bone regeneration.

Rationale: Osteoporotic patients suffer symptoms of excessive osteoclastogenesis and impaired osteogenesis, resulting in a great challenge to treat osteoporosis-related bone defects. Based on the positive effect of rare earth elements on bone metabolism and bone regeneration, we try to prove the hypothesis that the La3+ dopants in lanthanum-substituted MgAl layered double hydroxide (La-LDH) nanohybrid scaffolds simultaneously activate osteogenesis and inhibit osteoclastogenesis.Methods: A freeze-drying technology was employed to construct La-LDH nanohybrid scaffolds. The in vitro osteogenic and anti-osteoclastogenic activities of La-LDH nanohybrid scaffolds were evaluated by using ovariectomized rat bone marrow stromal cells (rBMSCs-OVX) and bone marrow-derived macrophages (BMMs) as cell models. The in vivo bone regeneration ability of the scaffolds was investigated by using critical-size calvarial bone defect model of OVX rats.Results: La-LDH nanohybrid scaffolds exhibited three-dimensional macroporous structure, and La-LDH nanoplates arranged perpendicularly on chitosan organic matrix. The La3+ dopants in the scaffolds promote proliferation and osteogenic differentiation of rBMSCs-OVX by activating Wnt/β-catenin pathway, leading to high expression of ALP, Runx-2, COL-1 and OCN genes. Moreover, La-LDH scaffolds significantly suppressed RANKL-induced osteoclastogenesis by inhibiting NF-κB signaling pathway. As compared with the scaffolds without La3+ dopants, La-LDH scaffolds provided more favourable microenvironment to induce new bone in-growth along macroporous channels.Conclusion: La-LDH nanohybrid scaffolds possessed the bi-directional regulation functions on osteogenesis and osteoclastogenesis for osteoporotic bone regeneration. The modification of La3+ dopants in bone scaffolds provides a novel strategy for osteoporosis-related bone defect healing.

Species-Specificity of Transcriptional Regulation and the Response to Lipopolysaccharide in Mammalian Macrophages.

Mammalian macrophages differ in their basal gene expression profiles and response to the toll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS). In human macrophages, LPS elicits a temporal cascade of transient gene expression including feed forward activators and feedback regulators that limit the response. Here we present a transcriptional network analysis of the response of sheep bone marrow-derived macrophages (BMDM) to LPS based upon RNA-seq at 0, 2, 4, 7, and 24 h post-stimulation. The analysis reveals a conserved transcription factor network with humans, and rapid induction of feedback regulators that constrain the response at every level. The gene expression profiles of sheep BMDM at 0 and 7 h post LPS addition were compared to similar data obtained from goat, cow, water buffalo, horse, pig, mouse and rat BMDM. This comparison was based upon identification of 8,200 genes annotated in all species and detected at >10TPM in at least one sample. Analysis of expression of transcription factors revealed a conserved transcriptional millieu associated with macrophage differentiation and LPS response. The largest co-expression clusters, including genes encoding cell surface receptors, endosome–lysosome components and secretory activity, were also expressed in all species and the combined dataset defines a macrophage functional transcriptome. All of the large animals differed from rodents in lacking inducible expression of genes involved in arginine metabolism and nitric oxide production. Instead, they expressed inducible transporters and enzymes of tryptophan and kynurenine metabolism. BMDM from all species expressed high levels of transcripts encoding transporters and enzymes involved in glutamine metabolism suggesting that glutamine is a major metabolic fuel. We identify and discuss transcripts that were uniquely expressed or regulated in rodents compared to large animals including ACOD1, CXC and CC chemokines, CD163, CLEC4E, CPM, CSF1, CSF2, CTSK, MARCO, MMP9, SLC2A3, SLC7A7, and SUCNR1. Conversely, the data confirm the conserved regulation of multiple transcripts for which there is limited functional data from mouse models and knockouts. The data provide a resource for functional annotation and interpretation of loci involved in susceptibility to infectious and inflammatory disease in humans and large animal species.

Switch of macrophage fusion competency by 3D matrices

Foreign body reaction reflects the integration between biomaterials and host cells. At the implantation microenvironment, macrophages usually fuse into multinuclear cells, also known as foreign body giant cells, to respond to the biomaterial implants. To understand the biomaterial-induced macrophage fusion, we examined whether biomaterial alone can initiate and control the fusion rate without exogenous cytokines and chemicals. We introduced a collagen-based 3D matrix to embed Raw264.7 cell line and primary rat bone marrow-derived macrophages. We found the biomaterial-stimuli interacted regional macrophages and altered the overall fusogenic protein expressions to regulate the macrophage fusion rate. The fusion rate could be altered by modulating the cell-matrix and cell-cell adhesions. The fused macrophage morphologies, the nuclei number in the fused macrophage, and the fusion rates were matrix dependent. The phenomena were also observed in the in vivo models. These results suggest that the biomaterial-derived stimuli exert similar functions as cytokines to alter the competency of macrophage fusion as well as their drug sensitivity in the biomaterial implanted tissue environment. Furthermore, this in vitro 3D-matrix model has the potential to serve as a toolbox to predict the host tissue response on implanted biomaterials.

RM-CSF efficiently replaces L929 in generating mouse and rat bone marrow-derived macrophages for in vitro functional studies of immunity to intracellular bacteria

Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as a prototype. We compared rM-CSF with L929 supernatant in terms of their effects on mouse and rat macrophage growth, maturation patterns, surface marker expression, and the expression of selected genes. Further, we compared macrophage infectivity and bacterial replication using LVS. Finally, we compared the in vitro function of BMDMs co-cultured with splenocytes from vaccinated animals in terms of their control of intramacrophage bacterial replication, as well as production of cytokines and nitric oxide. We demonstrated that rM-CSF produced BMDMs with similar, or minimal, phenotypic and gene expression outcomes compared to those generated with media containing L929 supernatant. Most importantly, functional outcomes were similar. Taken together, our data support the use of the rM-CSF in cell culture media as an alternative to L929-supplemented media for functional bioassays that use C57BL/6J mouse or Fischer 344 rat BMDMs to study intracellular infections. This comparison therefore facilitates future protocol standardization.

BabaoDan cures hepatic encephalopathy by decreasing ammonia levels and alleviating inflammation in rats.

BabaoDan (BBD) is a famous traditional Chinese formula frequently used in TCM clinics to eliminate jaundice and treat infectious viral hepatitis. This paper assesses BBD's preventive and therapeutic effects on hepatic encephalopathy after liver cirrhosis (CHE) and acute liver failure (AHE) in rats and explains its possible mechanism of action. CHE rat model was established by injection of carbon tetrachloride (CCl4) twice a week for a total of 9 weeks and then by injection of thioacetamide (TAA) to induce hepatic encephalopathy. AHE rat model was established by injection of TAA once a day for a total of 3 days. In CHE rat model, BBD was gavaged once a day at the end of the 6th week until the experiment ended. In AHE rat model,BBD was gavaged once a day 3 days before TAA injection until the experiment ended. The preventive and therapeutic effects of BBD on brain dysfunction, as well as liver injury, pathology and fibrosis were evaluated in vivo. The role of BBD in the regulation of inflammatory factors and myeloid differentiation factor 88/Toll-like receptor 4/nuclear factor kappa-B (TLR4/MyD88/NK-κ B) pathway was detected in both liver and brain in vivo. The rat bone marrow derived macrophages (BMDMs) were activated by Lipopolysaccharide (LPS), and the role of BBD in the regulation of inflammatory factors and NK-κ B pathway were detected in vitro. In CHE rat model: BBD significantly improved the total distance as well as the activity rate of rats. BBD also improved the learning and memory abilities of rats compared with the control group. In addition, BBD effectively decreased ammonia levels and significantly decreased the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBil) and total bile acid (TBA), as well as improved the levels of total protein (TP) and albumin (Alb). In the liver, BBD not only inhibited the gene expressions of tumor necrosis factor alpha (TNF-α), interleukini-6 (IL-6), TLR4, MyD88, and NF-κ B but also inhibited the protein expressions of TLR4, MyD88, NK-κ B and TNF-α. In the brain, BBD inhibited the gene expressions of iNOS, IL-6, TNF-α, TLR-4, MyD88, and NF-κ B, as well as inhibited the protein expressions of TLR4, MyD88, P65 TNF-α and ionized calcium binding adapter molecule 1 (Iba-1). BBD also decreased NO and TNF-α in the blood. BBD improved neurological scores, blood ammonia levels and the brain inflammatory gene expressions of iNOS, TNF-α and IL-1β. BBD also improved liver function biomarkers such as ALT, TBil, TBA, TP, ALB and inflammatory and apoptotic gene expressions of TNF-α, IL-1β, IL-6, Bax, Bcl-2, caspase-9, caspase-3 and NF-κ B. In LPS-activated rat BMDMs, BBD decreased NO and TNF-α production in BMDM culture supernatant. In addition, BBD inhibited the gene expressions of TNF-α, IL-1 β and IL-6 as well as the phosphorylation of P65. BBD can prevent and cure hepatic encephalopathy (HE) derived from both chronic and acute liver diseases. BBD can reduce hyperammonemia as well as the systematic and neurological inflammation. Inflammation is likely an important target of BBD to treat HE. The anti-inflammatory role of BBD may lie in its regulation of the TLR4/MyD88/NF-κ B pathways.

Adjuvant role of macrophages in stem cell-induced cardiac repair in rats

Bone marrow-derived mesenchymal stem cells (BMMSCs) are used extensively for cardiac repair and interact with immune cells in the damaged heart. Macrophages are known to be modulated by stem cells, and we hypothesized that priming macrophages with BMMSCs would enhance their therapeutic efficacy. Rat bone marrow-derived macrophages (BMDMs) were stimulated by lipopolysaccharide (LPS) with or without coculture with rat BMCs. In the LPS-stimulated BMDMs, induction of the inflammatory marker iNOS was attenuated, and the anti-inflammatory marker Arg1 was markedly upregulated by coculture with BMMSCs. Myocardial infarction (MI) was induced in rats. One group was injected with BMMSCs, and a second group was injected with MIX (a mixture of BMMSCs and BMDMs after coculture). The reduction in cardiac fibrosis was greater in the MIX group than in the BMC group. Cardiac function was improved in the BMMSC group and was substantially improved in the MIX group. Angiogenesis was better in the MIX group, and anti-inflammatory macrophages were more abundant in the MIX group than in the BMMSC group. In the BMMSCs, interferon regulatory factor 5 (IRF5) was exclusively induced by coculture with macrophages. IRF5 knockdown in BMMSCs failed to suppress inflammatory marker induction in the macrophages. In this study, we demonstrated the successful application of BMDMs primed with BMMSCs as an adjuvant to cell therapy for cardiac repair.

Upregulation of Enzymatic Idebenone Reduction Activity after Proinflammatory Stimulation of Microglia

Mitochondrial bioenergetic dysfunction is a component of nearly all neurodegenerative diseases. One strategy to overcome this dysfunction is to replace the loss of mitochondrial reducing equivalents using exogenous electron donors. The drug was recently approved in Europe for the treatment of Leber’s Hereditary Optic Neuropathy, a mitochondrial disease due to deficient Complex I activity. For efficacy, it is believed that idebenone requires enzymatic reduction to idebenol. Previously, quinone reduction activity was mostly attributed to NAD(P)H quinone oxidoreductases, particularly NQO1. In the brain, NQO1 is primarily expressed in astrocytes; we therefore hypothesized that astrocytes would display higher NAD(P)H-dependent idebenone reduction activity compared to other brain cell types. Using lysates derived from primary cells, we found that rat cortical astrocytes have the highest capacity for idebenone reduction, with little to no activity observed in rat cortical neurons or microglia. As expected, idebenone reduction in astrocytes was fully inhibited by the NQO1 inhibitor dicoumarol. Interestingly, we found a significant increase in idebenone reduction potential in lysates derived from microglia treated with the pro-inflammatory stimulus lipopolysaccharide (LPS) plus interferon-gamma (IFN-γ) for 18 hours. This activity was not sensitive to dicoumarol and was not observed in rat bone marrow-derived macrophages similarly stimulated with LPS/IFN-γ. Preliminary results suggest that it may be a multi-subunit flavoprotein. Identification of the enzyme responsible for this novel idebenone-reducing activity may lead to improved therapeutic strategies using idebenone as well as suggest other diseases wherein idebenone may have a beneficial effect.

Sphingosylphosphorylcholine inhibits macrophage adhesion to vascular smooth muscle cells

Inflammation in de-endothelialised arteries contributes to the development of cardiovascular diseases. The process that initiates this inflammatory response is the adhesion of monocytes/macrophages to exposed vascular smooth muscle cells, typically stimulated by cytokines such as tumour necrosis factor-α (TNF). The aim of this study was to determine the effect of the sphingolipid sphingosylphosphorylcholine (SPC) on the interaction of monocytes/macrophages with vascular smooth muscle cells. Rat aortic smooth muscle cells and rat bone marrow-derived macrophages were co-cultured using an in vitro assay following incubation with sphingolipids to assess inter-cellular adhesion. We reveal that SPC inhibits the TNF-induced adhesion of macrophages to smooth muscle cells. This anti-adhesive effect was the result of SPC-induced changes to the smooth muscle cells (but not the macrophages) and was mediated, at least partly, via the sphingosine 1-phosphate receptor subtype 2. Lipid raft domains were also required. Although SPC did not alter expression or membrane distribution of the adhesion proteins intercellular adhesion molecule-1 and vascular cellular adhesion protein-1 in smooth muscle cells, SPC preincubation inhibited the TNF-induced increase in inducible nitric oxide synthase (NOS2) resulting in a subsequent decrease in nitric oxide production. Inhibiting NOS2 activation in smooth muscle cells led to a decrease in the adhesion of macrophages to smooth muscle cells. This study has therefore delineated a novel pathway which can inhibit the interaction between macrophages and vascular smooth muscle cells via SPC-induced repression of NOS2 expression. This mechanism could represent a potential drug target in vascular disease.