Regulates Macrophage Migration Research Articles

Plexin D1 negatively regulates macrophage-derived foam cell migration via the focal adhesion kinase/Paxillin pathway

BackgroundMacrophage-derived foam cell formation is a hallmark of atherosclerosis and is retained during plaque formation. Strategies to inhibit the accumulation of these cells hold promise as viable options for treating atherosclerosis. Plexin D1 (PLXND1), a member of the Plexin family, has elevated expression in atherosclerotic plaques and correlates with cell migration; however, its role in macrophages remains unclear. We hypothesize that the guidance receptor PLXND1 negatively regulating macrophage mobility to promote the progression of atherosclerosis. MethodsWe utilized a mouse model of atherosclerosis based on a high-fat diet and an ox-LDL- induced foam cell model to assess PLXND1 levels and their impact on cell migration. Through western blotting, Transwell assays, and immunofluorescence staining, we explored the potential mechanism by which PLXND1 mediates foam cell motility in atherosclerosis. ResultsOur study identifies a critical role for PLXND1 in atherosclerosis plaques and in a low-migration capacity foam cell model induced by ox-LDL. In the aortic sinus plaques of ApoE−/− mice, immunofluorescence staining revealed significant upregulation of PLXND1 and Sema3E, with colocalization in macrophages. In macrophages treated with ox-LDL, increased expression of PLXND1 led to reduced pseudopodia formation and decreased migratory capacity. PLXND1 is involved in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK. Additionally, FAK inhibitors counteract the ox-LDL-induced migration suppression by modulating the phosphorylation states of FAK, Paxillin and their downstream effectors CDC42 and PAK. ConclusionOur findings indicate that PLXND1 plays a role in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK to promoting atherosclerosis.

Paracrine Ovarian Cancer Cell-Derived CSF1 Signaling Regulates Macrophage Migration Dynamics in a 3D Microfluidic Model that Recapitulates In Vivo Infiltration Patterns in Patient-Derived Xenografts.

A high density of macrophages in the ovarian cancer microenvironment is associated with disease progression and poor outcomes. Understanding cancer-macrophage interaction mechanisms that establish this pro-tumorigenic microenvironment is critical for developing macrophage-targeted therapies. Here, 3D microfluidic assays and patient-derived xenografts are utilized to define the role of cancer-derived colony stimulating factor 1 (CSF1) on macrophage infiltration dynamics toward ovarian cancer cells. It is demonstrated that multiple ovarian cancer models promote the infiltration of macrophages into a 3D extracellular matrix in vitro in a cell density-dependent manner. Macrophages exhibit directional migration and increased migration speed under both direct interactions with cancer cells embedded within the matrix and paracrine crosstalk with cancer cells seeded in an independent microchannel. It is also found that platinum-based chemotherapy increases macrophage recruitment and the levels of cancer cell-derived CSF1. Targeting CSF1 signaling under baseline or chemotherapy-treatment conditions reduces the number of infiltrated macrophages. It is further shown that results obtained with the 3D microfluidic model reflect the recruitment profiles of macrophages in patient-derived xenografts in vivo. These findings highlight the role of CSF1 signaling in establishing macrophage-rich ovarian cancer microenvironments, as well as the utility of microfluidic models in recapitulating 3D tumor ecosystems and dissecting cancer-macrophage signaling.

Moxibustion of Zusanli (ST36) and Shenshu (BL23) alleviates the inflammation of rheumatoid arthritis in rats through regulating macrophage migration inhibitory factor/glucocorticoids signaling.

To test the hypothesis that moxibustion may inhibit rheumatoid arthritis (RA) synovial inflammation by regulating the expression of macrophage migration inhibitory factor (MIF)/glucocorticoids (GCs). Fifty male Sprague-Dawley rats were randomly divided into five groups (n = 10 each): blank Control (CON) group, RA Model (RA) group, Moxibustion (MOX) group, MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) group, and Moxibustion + MIF inhibitor ISO-1 (MOX + ISO-1) group. Rats in the ISO-1 group and ISO-1 + MOX group were intraperitoneally injected with the inhibitor ISO-1. The rats in the RA group, ISO-1 group, MOX group, and ISO-1 + MOX group were injected with Freund's complete adjuvant (FCA) in the right hind footpad to establish an experimental RA rat model. In the MOX group and MOX + ISO-1 group, rats were treated with Moxa. The thickness of the footpads of the rats in each group was measured at three-time points before, after modeling and after moxibustion treatment. The contents of serum MIF, corticosterone (CORT), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by enzyme-linked immunosorbent assay; and the contents of synovial MIF were detected by Western blot. Hematoxylin-eosin (HE) staining method was used to observe the pathological changes of synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease. Moxibustion was found to reduce the level of MIF and alleviate inflammation in RA rats in this study. In addition, after inhibiting the expression of MIF, the level of CORT increased, and the level of TNF-α decreased. Treating RA rats with inhibited MIF by moxibustion, the level of CORT was almost unchanged, but the level of TNF-α further decreased. The correlation analysis data suggested that MIF was positively related to the expression of TNF-α and negatively correlated with the expression of CORT. Reducing MIF to increase CORT and decrease TNF-α by moxibustion treatment in RA. MIF may be a factor for moxibustion to regulate the expression of CORT, but the expression of TNF-α is due to the incomplete regulation of the MIF. This study added to the body of evidence pointing to moxibustion's anti-inflammatory mechanism in the treatment of RA.

Repolarization of Immunosuppressive Macrophages by Targeting SLAMF7-Regulated CCL2 Signaling Sensitizes Hepatocellular Carcinoma to Immunotherapy.

CCL2 upregulation caused by SLAMF7 deficiency in hepatocellular carcinoma cells induces immunosuppressive macrophage polarization and confers resistance to immune checkpoint blockade, providing potential biomarkers and targets to improve immunotherapy response in patients.

TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus.

Aspergillus fumigatus can infect immunocompromised individuals and cause chronic and fatal invasive fungal infections. A better understanding of the molecular mechanisms of A. fumigatus-host interactions may provide new references for disease treatment. In this study, we demonstrated that the TRAF3 gene plays an important role in the early infection of A. fumigatus by regulating the resistance of lung epithelial cells to A. fumigatus. Macrophages are the most abundant innate immune cells in the alveoli; however, few studies have reported on the interactions between lung epithelial cells and macrophages in response to A. fumigatus invasion. In our study, it was demonstrated that the TRAF3 gene reduces migration to macrophages and cytokine production by negatively regulating lung epithelial cell adhesion and internalization of A. fumigatus spores. Together, our results provide new insights into lung epithelial cell-macrophage interactions during A. fumigatus infection.

MiR-186-5p targets TGFβR2 to inhibit RAW264.7 cell migration and proliferation during mouse skin wound healing.

Active peptides play a vital role in the development of new drugs and the identification and discovery of drug targets. As the first reported native peptide homodimer with pro-regenerative potency, OA-GP11d could potentially be used as a novel molecular probe to help elucidate the molecular mechanism of skin wound repair and provide new drug targets. Bioinformatics analysis and luciferase assay were adopted to determine microRNAs (miRNAs) and its target. The prohealing potency of the miRNA was determined by MTS and a Transwell experiment against mouse macrophages. Enzyme-linked immunosorbent assay, realtime polymerase chain reaction, and western blotting were performed to explore the molecular mechanisms. In this study, OA-GP11d was shown to induce Mus musculus microRNA-186-5p (mmu-miR-186-5p) down-regulation. Results showed that miR-186-5p had a negative effect on macrophage migration and proliferation as well as a targeted and negative effect on TGF-β type II receptor (TGFβR2) expression and an inhibitory effect on activation of the downstream SMAD family member 2 (Smad2) and protein-p38 kinase signaling pathways. Importantly, delivery of a miR-186-5p mimic delayed skin wound healing in mice. miR-186-5p regulated macrophage migration and proliferation to delay wound healing through the TGFβR2/Smad2/p38 molecular axes, thus providing a promising new pro-repair drug target.

Identification of key pathways in zirconia/dental pulp stem cell composite scaffold-mediated macrophage polarization through transcriptome sequencing

ABSTRACT Seed cells and scaffold materials are essential components of tissue engineering. In this study, we investigated the key pathway of the zirconia/dental pulp stem cell composite scaffold in regulating macrophage polarization by transcriptome sequencing. We established N-rGO/ZrO2 composite scaffold and confirmed its structure using various analytical techniques, including SEM, TEM, FTIR, Raman spectra, XPS, and XRD. DPSCs were seeded onto N-rGO/ZrO2 composite scaffold material, and their proliferation, adhesion, and osteogenic differentiation were evaluated by CCK-8, immunofluorescence staining, ALP staining, and alizarin red staining. We then co-cultured DPSCs combined with N-rGO/ZrO2 as composite material with THP-1 cells in a transwell system to investigate the effect of the composite on macrophage polarization. The levels of pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes were assessed by RT-qPCR and western blot. Through bulk RNA sequencing, we detected the transcriptional characteristics of macrophages under the regulation of the composite materials, and identified the differential genes using the DEseq2 package. We also analyzed the cellular and molecular functions of differentially expressed genes (DEGs) in THP-1 cells with DPSCs combined with N-rGO/ZrO2 treatment using GO enrichment analysis and KEGG pathway enrichment analysis. Our results showed that N-rGO/ZrO2 composite scaffold promoted the proliferation, adhesion, and osteogenic differentiation of DPSCs. Moreover, N-rGO/ZrO2 composite scaffold combined with DPSCs regulated macrophage migration, polarization, and glycolysis. Mechanistically, the combination of N-rGO/ZrO2 composite materials and DPSCs regulated macrophage polarization by activating the TNF signaling pathway. This finding provides a new approach to the clinical preservation of maxillofacial bone defect repair.

CX3CL1/CX3CR1 interaction protects against lipotoxicity-induced nonalcoholic steatohepatitis by regulating macrophage migration and M1/M2 status

Background and objectivesChemokine (C-X3-C motif) ligand 1 (CX3CL1) and its receptor CX3CR1 regulate the migration and activation of immune cells and are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the mechanism remains elusive. Here, the roles of CX3CL1/CX3CR1 in the macrophage migration and polarization in the livers of NASH mice were investigated. Methods and resultsThe expression of Cx3cl1 and Cx3cr1 was markedly upregulated in the livers of lipotoxicity-induced NASH mice. CX3CR1 was predominantly expressed by F4/80+ macrophages and to a lesser degree by hepatic stellate cells or endothelial cells in the livers of NASH mice. Flow cytometry analysis revealed that, compared with chow-fed mice, NASH mice exhibited a significant increase in CX3CR1+ expression by liver macrophages (LMs), particularly M1 LMs. CX3CR1 deficiency caused a significant increase in inflammatory monocyte/macrophage infiltration and a shift toward M1 dominant macrophages in the liver, thereby exacerbating the progression of NASH. Moreover, transplantation of Cx3cr1−/− bone marrow was sufficient to cause glucose intolerance, inflammation, and fibrosis in the liver. In addition, deletion of CCL2 in Cx3cr1−/− mice alleviated NASH progression by decreasing macrophage infiltration and inducing a shift toward M2 dominant LMs. Importantly, overexpression of CX3CL1 in vivo protected against hepatic fibrosis in NASH. ConclusionPharmacological therapy targeting liver CX3CL1/CX3CR1 signaling might be a candidate for the treatment of NASH.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Myometrial-derived CXCL12 promotes lipopolysaccharide induced preterm labour by regulating macrophage migration, polarization and function in mice.

Preterm birth is a major contributor to neonatal mortality and morbidity. Infection results in elevation of inflammation‐related cytokines followed by infiltration of immune cells into gestational tissue. CXCL12 levels are elevated in preterm birth indicating it may have a role in preterm labour (PTL); however, the pathophysiological correlations between CXCL12/CXCR4 signalling and premature labour are poorly understood. In this study, PTL was induced using lipopolysaccharide (LPS) in a murine model. LPS induced CXCL12 RNA and protein levels significantly and specifically in myometrium compared with controls (3‐fold and 3.5‐fold respectively). Highest levels were found just before the start of labour. LPS also enhanced the infiltration of neutrophils, macrophages and T cells, and induced macrophage M1 polarization. In vitro studies showed that condition medium from LPS‐treated primary smooth muscle cells (SMC) induced macrophage migration, M1 polarization and upregulated inflammation‐related cytokines such as interleukin (IL)‐1, IL‐6 and tumor necrosis factor alpha (TNF‐α). AMD3100 treatment in pregnant mice led to a significant decrease in the rate of PTL (70%), prolonged pregnancy duration and suppressed macrophage infiltration into gestation tissue by 2.5‐fold. Further, in‐vitro treatment of SMC by AMD3100 suppressed the macrophage migration, decreased polarization and downregulated IL‐1, IL‐6 and TNF‐α expression. LPS treatment in pregnant mice induced PTL by increasing myometrial CXCL12, which recruits immune cells that in turn produce inflammation‐related cytokines. These effects stimulated by LPS were completely reversed by AMD3100 through blocking of CXCL12/CXCR4 signalling. Thus, the CXCL12/CXCR4 axis presents an excellent target for preventing infection and inflammation‐related PTL.

EZH2 Might Affect Macrophage Chemotaxis and Anti-Inflammatory Factors by Regulating CCL2 in Dental Pulp Inflammation.

Objectives We aimed to evaluate the effects of Enhancer of Zeste Homolog 2 (EZH2) on regulation of macrophage migration and expression of anti-inflammatory genes in pulpitis. Methods Dental pulp inflammation was verified by histology in rat pulpitis model induced by lipopolysaccharide (LPS). Immunohistochemistry staining was used to detect changes of the expression of EZH2 and tumor necrosis factor alpha (TNF-α) in dental pulp inflammation. The expression of EZH2, CCL2, and cluster of differentiation 68 (CD68: macrophage surface marker) was measured by immunofluorescence staining. The effect of EZH2 on microphage migration was assessed by cell migration assay. The expressions of anti-inflammatory cytokine interleukins (IL-4 and IL-10) and transforming growth factor-β (TGF-β) in HDPCs which were treated by EZH2 complex, CCL2 complex, and CCL2 antibody were examined by quantitative real-time polymerase chain reaction (q-PCR). Results The expression of TNF-α gradually increased in dental pulp inflammation. The expression of EZH2 in dental pulp decreased in 8 hours after LPS stimulation. However, the expression of EZH2 gradually increased in dental pulp after 1 day stimulation by LPS. The results of immunofluorescence staining showed that the expressions of EZH2, CCL2, and CD68 were significantly upregulated in dental pulp inflammation of rats. EZH2 could enhance macrophage migration. And the chemotactic activity of macrophages exposed to supernatants of EZH2-treated HDPCs could be inhibited by CCL2 inhibition. In addition, EZH2 suppressed the expression of anti-inflammatory genes, but CCL2 inhibition reversed the downregulation of anti-inflammatory factors, including IL-4 and TGF-β in HDPCs. Conclusions EZH2 might affect chemotaxis of macrophages and the expression of anti-inflammatory factors by regulating CCL2. EZH2 plays an important role in the development of dental pulp inflammation, and it might be as a target for treatment of pulpitis.

MicroRNA-409-3p regulates macrophage migration in polymyositis through targeting CXCR4

Background Macrophage migration and infiltration contribute to the pathogenesis of polymyositis (PM). This study aims to investigate the effect and underlying mechanism of miR-409-3p on macrophage migration in PM. Methods The GSE143845 database was used to predict the altered expression of microRNAs (miRNAs) in PM. The quantitative real-time PCR (qRT-PCR), western blot and Transwell assay were performed to detect migration of macrophages and expressions of related molecules. A luciferase activity assay was conducted to confirm the binding of miR-409-3p and CXCR4 3'-UTR. Next, a mouse model of experimental autoimmune myositis (EAM) was established. Haematoxylin and eosin (HE) staining, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) were used to measure associated factors. Results MiR-409-3p was downregulated in PM of GSE143845 database and patients. Differently, the serum creatine kinase (s-CK), TNF-α, and IL-6 in patients with PM were increased. Furthermore, miR-409-3p mimic transfection reduced the migration of macrophages and CXCR4 levels, while miR-409-3p inhibitor exerted the opposite effects. CXCR4 was a target of miR-409-3p, and the effect of CXCR4 on promoting macrophage migration was reversed by miR-409-3p mimic. In vivo, miR-409-3p agomir injection reduced inflammatory cells, macrophages, and TNFα and IL-6 levels in muscles and serum of EAM mouse models. Conclusions In conclusion, miR-409-3p reduces the migration of macrophages through negatively regulating CXCR4 expression in PM.

The Epidermal Growth Factor Ligand Spitz Modulates Macrophage Efferocytosis, Wound Responses and Migration Dynamics During Drosophila Embryogenesis.

How multifunctional cells such as macrophages interpret the different cues within their environment and undertake an appropriate response is a key question in developmental biology. Understanding how cues are prioritized is critical to answering this – both the clearance of apoptotic cells (efferocytosis) and the migration toward damaged tissue is dependent on macrophages being able to interpret and prioritize multiple chemoattractants, polarize, and then undertake an appropriate migratory response. Here, we investigate the role of Spitz, the cardinal Drosophila epidermal growth factor (EGF) ligand, in regulation of macrophage behavior in the developing fly embryo, using activated variants with differential diffusion properties. Our results show that misexpression of activated Spitz can impact macrophage polarity and lead to clustering of cells in a variant-specific manner, when expressed either in macrophages or the developing fly heart. Spitz can also alter macrophage distribution and perturb apoptotic cell clearance undertaken by these phagocytic cells without affecting the overall levels of apoptosis within the embryo. Expression of active Spitz, but not a membrane-bound variant, can also increase macrophage migration speeds and impair their inflammatory responses to injury. The fact that the presence of Spitz specifically undermines the recruitment of more distal cells to wound sites suggests that Spitz desensitizes macrophages to wounds or is able to compete for their attention where wound signals are weaker. Taken together these results suggest this molecule regulates macrophage migration and their ability to dispose of apoptotic cells. This work identifies a novel regulator of Drosophila macrophage function and provides insights into signal prioritization and integration in vivo. Given the importance of apoptotic cell clearance and inflammation in human disease, this work may help us to understand the role EGF ligands play in immune cell recruitment during development and at sites of disease pathology.

Evidence for the loss of plasminogen receptor KT gene in chicken.

The loss of conserved genes has the potential to alter phenotypes drastically. Screening of vertebrate genomes for lineage-specific gene loss events has identified numerous natural knockouts associated with specific phenotypes. We provide evidence for the loss of a multi-exonic plasminogen receptor KT (PLGRKT) protein-encoding gene located on the Z chromosome in chicken. Exons 1 and 2 are entirely missing; remnants of exon 3 and a mostly intact exon 4 are identified in an assembly gap-free region in chicken with conserved synteny across species and verified using transcriptome and genome sequencing. PLGRKT gene disrupting changes are present in representative species from all five galliform families. In contrast to this, the presence of an intact transcriptionally active PLGRKT gene in species such as mallard, swan goose, and Anolis lizard suggests that gene loss occurred in the galliform lineage sometime between 68 and 80Mya. The presence of galliform specific chicken repeat 1 (CR1) insertion at the erstwhile exon 2 of PLGRKT gene suggests repeat insertion-mediated loss. However, at least nine other independent PLGRKT coding frame disrupting changes in other bird species are supported by genome sequencing and indicate a role for relaxed purifying selection before CR1 insertion. The recurrent loss of a conserved gene with a role in the regulation of macrophage migration, efferocytosis, and blood coagulation is intriguing. Hence, we propose potential candidate genes that might be compensating the function of PLGRKT based on the presence of a C-terminal lysine residue, transmembrane domains, and gene expression patterns.

Asap1 Affects the Susceptibility of Zebrafish to Mycobacterium by Regulating Macrophage Migration.

The ADP ribosylation factor (ARF) GTPase activation protein ASAP1 possesses multiple biological functions, including regulation of cytoskeletal dynamics, small GTP-binding protein receptor recycling, and intracellular vesicle trafficking. Recently, ASAP1 polymorphisms have been reported to be associated with human susceptibility to tuberculosis (TB) according to a large-scale genome-wide association study (GWAS); ASAP1 expression affects dendritic cell migration, which may be involved in TB predisposition. However, it remains unclear whether ASAP1 affects TB in vivo. To address this issue, we used zebrafish as a model system to examine the effects of Asap1 against Mycobacterium marinum, an organism closely related to Mycobacterium tuberculosis. Two zebrafish asap1 homologs (asap1a and asap1b) were identified and characterized. By morpholino knockdown of asap1a and asap1b as a whole, we found that the asap1 morphants showed a higher mycobacterial load than the controls, which was almost rescued by injecting asap1 mRNA that confers resistance to mycobacterial infection. These Asap1-depleted zebrafish also exhibited decreased macrophage migration in response to tail injury or upon infection with M. marinum in the hindbrain ventricle, which was also proved in THP1-derived macrophages of knockdown ASAP1. Together, these findings represent a new perspective on the role of Asap1 in resistance to mycobacterial infection.

2128-P: 12/15-Lipoxygenase Regulates Macrophage Migration during Islet Inflammation

Pancreatic islet inflammation is a shared characteristic of type 1 and type 2 diabetes pathogenesis, and macrophage infiltration has been shown to exacerbate this inflammation. The enzyme 12/15-Lipoxygenase (12/15-LOX) which is involved in polyunsaturated fatty acid metabolism, has also been implicated in islet inflammation. Based on its established roles in cellular migration in other contexts, we hypothesized that 12/15-LOX promotes macrophage infiltration. To test this hypothesis, we used two complementary model systems: zebrafish and mice. First, we knocked down 12/15-LOX in Tg(ins:NTR) zebrafish with an antisense morpholino. When these zebrafish are treated with the prodrug metronidazole (MTZ), β cells are specifically injured, and macrophages accumulate in the islet. After a 6-hour incubation with MTZ, we observed a significant reduction in the number of infiltrating macrophages in the islets of 12/15-LOX knockdown larvae as compared to control larvae (2.7±0.8 vs. 1.17±0.7, P<0.05). Similarly, injured β cells in zebrafish treated with the 12/15-LOX inhibitor ML355 showed a significant reduction in the number of islet-infiltrating macrophages compared to vehicle-treated larvae (3.67±0.7 vs. 1.2±0.3, P<0.05). Next, using transwell assays, we measured the migration of peritoneal macrophages that were isolated from 12/15-LOX knockout mice in media that was conditioned by islet culture. Again, we observed a significant reduction in macrophage migration with 12/15-LOX deficiency compared to controls (162.8±15.5 vs. 108.4±15.1, P<0.05). Mechanistically, we found that the expression of the chemokine receptor CXCR3 was significantly downregulated in 12/15-LOX-knockout macrophages. Taken together, our data reveal a role for 12/15-LOX in macrophage migration that is conserved across species. Importantly, our study indicates that the signaling pathways downstream from 12/15-LOX are potential therapeutic targets for the resolution of inflammation-induced damage in the islets. Disclosure A. Kulkarni: None. A. Pineros: None. S. Ibrahim: None. M. Hernandez-Perez: None. S.A. Tersey: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. R.M. Anderson: None.

CXCR4-overexpressing Mesenchymal Stem Cells exhibit increased cell migration and cytokine secretion

Background & Aim The regenerative and immunomodulatory properties of Mesenchymal Stem Cells (MSCs) have been demonstrated extensively in vitro, and in vivo in various types of pre-clinical models. However, in clinical settings, while the safety profile of MSCs has been well-established, the efficacy data obtained in pre-clinical studies hasn't been recapitulated in most cases where MSCs have been administered to human subjects. Many factors are thought to hinder MSC-based therapies; low potency and retention, and limited homing to the site of injury. In the current study, we sought to engineer bone-marrow-derived MSCs (BMSCs) with improved migratory properties so as to facilitate homing to the site of injury. Methods, Results & Conclusion We utilized a genetic approach in which BMSCs were transfected with CXCR4 mRNA. CXCR4, a G-protein coupled receptor highly expressed on the surface of multiple cell types is best known for its role in hematopoiesis and leukocyte chemotaxis. We showed that transfection of CXCR4 mRNA into BMSCs for eight hours resulted in over 60% of cells positive for CXCR4 protein expression, as determined by flow cytometry. Less than 1% of un-transfected BMSCs expresses CXCR4 on their surface. The expression of CXCR4 was maintained post-thaw when transfected BMSCs were recovered from LN2 Storage. Transfected cells also maintained their viability post-thaw. In terms of functionality, we demonstrated that BMSCs expressing CXCR4 were more migratory compared to their naive counterparts lacking CXCR4. This effect on migration was augmented when CXCR4-BMSCs were induced to migrate towards SDF-1α, the cognate ligand for CXCR4. In addition to the effects on directed cell migration, with the use of a membrane-based antibody array, we showed that compared to control BMSCs, CXCR4-BMSCs secrete greater quantities of MCP-1, a key chemokine that regulates macrophage migration and infiltration. In summary, we have shown that transfection of BMSCs with CXCR4 mRNA increases directed cell migration and alters cytokine secretion. This is a potential strategy for generating BMSCs with superior homing properties for use in clinical settings.

Blockage of Kv1.3 regulates macrophage migration in acute liver injury by targeting δ-catenin through RhoA signaling.

Background: Activation of macrophages and infiltration are key events in acute liver injury (ALI). Kv1.3 plays an important role in regulating immunologic functions of macrophages and is extensively recognized as a potential ion channel for immunological diseases.Objective: We hypothesized that blockage of Kv1.3 may influence ALI by inhibiting macrophages infiltration in damaged liver tissues.Methods: Margatoxin was administered into the peritoneal cavity of ALI mice. The impact of this treatment on ALI and macrophage migration in vivo and in vitro was determined using immunohistochemistry, transwell migration, and wound healing assays.Results: MgTX treatment alleviated ALI in mice, as evidenced by reduced macrophage infiltration in liver tissues and lower serum levels of liver ALT and AST. RNA-seq profiling analysis showed that the most obvious change by MgTX treatment was downregulation of δ-catenin, a protein known to be associated with macrophage migration. The effect of MgTX on macrophage migration and involvement of δ-catenin was confirmed by transwell and wound healing assays. Overexpression of δ-catenin in RAW264.7 cells promoted migration, an event that was suppressed upon silencing of δ-catenin. Mechanistically, the expression of RhoA was regulated by the overexpression or knockdown of δ-catenin.Conclusion: These findings suggest a role for blockage of Kv1.3 channel in macrophage migration and reveal a new target in the treatment of ALI.

MicroRNA-21 deficiency attenuated atherogenesis and decreased macrophage infiltration by targeting Dusp-8

Background and aimsAtherosclerosis is a chronic inflammatory disorder mediated by macrophage activation. MicroRNA-21 (miR-21) is a key regulator in the macrophage inflammatory response. However, the functional role of miR-21 in atherogenesis is far from clear. Methods and resultsHere, we report that miR-21 is significantly upregulated in mouse atherosclerotic plaques and peripheral monocytes from patients with coronary artery disease. Compared with miR-21+/+apoE−/− mice (apoE−/− mice), miR-21−/−apoE−/− (double knockout, DKO) mice showed less atherosclerotic lesions, reduced presence of macrophages, decreased smooth muscle cells(SMC) and collagen content in the aorta. We further explored the role of miR-21 in macrophage activation in vitro. Bone marrow-derived macrophages (BMDMs) from DKO mice not only exhibit impaired function of migration induced by chemokine (C–C motif) ligand 2 (CCL2) but also a weakened macrophage-endothelium interaction activated by tumor necrosis factor-α (TNF-α). However, atherogenic inflammatory cytokine secretion was not affected by miR-21 in vitro or in vivo. Additionally, miR-21 knockdown in BMDMs directly derepressed the expression of dual specificity protein phosphatase 8 (Dusp-8), a previously validated miR-21 target in cardiac fibroblasts, which negatively regulates mitogen-activated protein kinase (MAPK) signaling, particularly the p38-and c-Jun N-terminal kinase (JNK)-related signaling pathways. ConclusionsThese data demonstrate that inhibition of miR-21 may restrict the formation of atherosclerotic plaques partly by regulating macrophage migration and adhesion, while, reduced SMCs and collagen content in plaques may lead to a less stable phenotype with the progression of atherosclerosis. Thus, the absence of miR-21 reduces atherosclerotic lesions but may not represent all benefit in atherosclerosis development.

Frontline Science: Antagonism between regular and atypical Cxcr3 receptors regulates macrophage migration during infection and injury in zebrafish.

The CXCR3‐CXCL11 chemokine‐signaling axis plays an essential role in infection and inflammation by orchestrating leukocyte trafficking in human and animal models, including zebrafish. Atypical chemokine receptors (ACKRs) play a fundamental regulatory function in signaling networks by shaping chemokine gradients through their ligand scavenging function, while being unable to signal in the classic G‐protein‐dependent manner. Two copies of the CXCR3 gene in zebrafish, cxcr3.2 and cxcr3.3, are expressed on macrophages and share a highly conserved ligand‐binding site. However, Cxcr3.3 has structural characteristics of ACKRs indicative of a ligand‐scavenging role. In contrast, we previously showed that Cxcr3.2 is an active CXCR3 receptor because it is required for macrophage motility and recruitment to sites of mycobacterial infection. In this study, we generated a cxcr3.3 CRISPR‐mutant to functionally dissect the antagonistic interplay among the cxcr3 paralogs in the immune response. We observed that cxcr3.3 mutants are more susceptible to mycobacterial infection, whereas cxcr3.2 mutants are more resistant. Furthermore, macrophages in the cxcr3.3 mutant are more motile, show higher activation status, and are recruited more efficiently to sites of infection or injury. Our results suggest that Cxcr3.3 is an ACKR that regulates the activity of Cxcr3.2 by scavenging common ligands and that silencing the scavenging function of Cxcr3.3 results in an exacerbated Cxcr3.2 signaling. In human, splice variants of CXCR3 have antagonistic functions and CXCR3 ligands also interact with ACKRs. Therefore, in zebrafish, an analogous regulatory mechanism appears to have evolved after the cxcr3 gene duplication event, through diversification of conventional and atypical receptor variants.