Macrophage Spreading Research Articles

Role of TRPV4 mechanosensing in foreign body response and giant cell formation

Implantable biomaterials and medical devices are used in millions of procedures each year worldwide. However, the implantation of these devices leads to the development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Despite decades of research, the mechanisms underlying the FBR remain poorly understood, as neither the materials from which these implants are made nor their chemical properties can explain triggering of the FBR. Hallmarks of the FBR include activation of macrophages at the tissue‐implant interface, formation of destructive foreign body giant cells (FBGCs), and development of fibrous tissue that encapsulates the implant. Here, we report that genetic ablation of TRPV4, an ion channel in the transient receptor potential vanilloid family, and which is a mechanosensor, is possibly the mediator of FBR. Specifically, we found that: 1) Trpv4 deletion in mice prevented macrophage accumulation, FBGC formation, and collagen accumulation in a subcutaneous implantation model; 2) the severity of the in vivo macrophage accumulation at the tissue‐implant interface was dependent on the stiffness of the implant; 3) genetic ablation of TRPV4 blocked macrophage adhesion and spreading on stiff matrix in bone marrow derived macrophages; and 4) genetic deficiency of TRPV4 blocked cytokine‐induced FBGC formation, which was restored by lentivirus‐mediated TRPV4 reintroduction. Furthermore, we found that: 5) TRPV4 activity (calcium influx) was augmented in response to a biomechanical stimulus; and 6) loss of TRPV4 activity reduced internal stiffness in macrophages as determined by Atomic Force Microscopy analysis. Taken together, these results suggest an important, previously unknown role for TRPV4 in FBR and giant cell formation.Support or Funding InformationFunding: This work was supported by NIH (1R01EB024556‐01) and NSF (CMMI‐1662776) grants to Shaik O. Rahaman.

Tuberculosis-associated IFN-I induces Siglec-1 on tunneling nanotubes and favors HIV-1 spread in macrophages.

While tuberculosis (TB) is a risk factor in HIV-1-infected individuals, the mechanisms by which Mycobacterium tuberculosis (Mtb) worsens HIV-1 pathogenesis remain scarce. We showed that HIV-1 infection is exacerbated in macrophages exposed to TB-associated microenvironments due to tunneling nanotube (TNT) formation. To identify molecular factors associated with TNT function, we performed a transcriptomic analysis in these macrophages, and revealed the up-regulation of Siglec-1 receptor. Siglec-1 expression depends on Mtb-induced production of type I interferon (IFN-I). In co-infected non-human primates, Siglec-1 is highly expressed by alveolar macrophages, whose abundance correlates with pathology and activation of IFN-I/STAT1 pathway. Siglec-1 localizes mainly on microtubule-containing TNT that are long and carry HIV-1 cargo. Siglec-1 depletion decreases TNT length, diminishes HIV-1 capture and cell-to-cell transfer, and abrogates the exacerbation of HIV-1 infection induced by Mtb. Altogether, we uncover a deleterious role for Siglec-1 in TB-HIV-1 co-infection and open new avenues to understand TNT biology.

Control of Matrix Stiffness Using Methacrylate-Gelatin Hydrogels for a Macrophage-Mediated Inflammatory Response.

The successful tissue integration of a biomedical material is mainly determined by the inflammatory response after implantation. Macrophage behavior toward implanted materials is pivotal to determine the extent of the inflammatory response. Hydrogels with different properties have been developed for various biomedical applications such as wound dressings or cell-loaded scaffolds. However, there is limited investigation available on the effects of hydrogel mechanical properties on macrophage behavior and the further host inflammatory response. To this end, methacrylate-gelatin (GelMA) hydrogels were selected as a model material to study the effect of hydrogel stiffness (2, 10, and 29 kPa) on macrophage phenotype in vitro and the further host inflammatory response in vivo. Our data showed that macrophages seeded on stiffer surfaces tended to induce macrophages toward a proinflammatory (M1) phenotype with increased macrophage spreading, more defined F-actin and focal adhesion staining, and more proinflammatory cytokine secretion and cluster of differentiation (CD) marker expression compared to those on surfaces with a lower stiffness. When these hydrogels were further subcutaneously implanted in mice to assess their inflammatory response, GelMA hydrogels with a lower stiffness showed more macrophage infiltration but thinner fibrotic capsule formation. The more severe inflammatory response can be attributed to the higher percentage of M1 macrophages induced by GelMA hydrogels with a higher stiffness. Collectively, our data demonstrated that macrophage behavior and the further inflammatory response are mechanically regulated by hydrogel stiffness. The macrophage phenotype rather than the macrophage number predominately determined the inflammatory response after the implantation, which can provide new insights into the future design and application of novel hydrogel-based biomaterials.

Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages.

HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is a restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR's normal role, which is to trap and destroy mannose-expressing pathogens.

Adhesion, motility and matrix-degrading gene expression changes in CSF-1-induced mouse macrophage differentiation.

Migratory macrophages play critical roles in tissue development, homeostasis and disease, so it is important to understand how their migration machinery is regulated. Whole-transcriptome sequencing revealed that CSF-1-stimulated differentiation of bone marrow-derived precursors into mature macrophages is accompanied by widespread, profound changes in the expression of genes regulating adhesion, actin cytoskeletal remodeling and extracellular matrix degradation. Significantly altered expression of almost 40% of adhesion genes, 60-86% of Rho family GTPases, their regulators and effectors and over 70% of extracellular proteases occurred. The gene expression changes were mirrored by changes in macrophage adhesion associated with increases in motility and matrix-degrading capacity. IL-4 further increased motility and matrix-degrading capacity in mature macrophages, with additional changes in migration machinery gene expression. Finally, siRNA-induced reductions in the expression of the core adhesion proteins paxillin and leupaxin decreased macrophage spreading and the number of adhesions, with distinct effects on adhesion and their distribution, and on matrix degradation. Together, the datasets provide an important resource to increase our understanding of the regulation of migration in macrophages and to develop therapies targeting disease-enhancing macrophages.

Positive role of calcium phosphate ceramics regulated inflammation in the osteogenic differentiation of mesenchymal stem cells.

Recently, researches have confirmed the crucial role of inflammatory response in Ca-P ceramic-induced osteogenesis, however, the underlying mechanism has not yet been fully understood. In this study, BCP and β-TCP ceramics were used as material models to investigate the effect of physicochemical properties on inflammatory response in vitro. The results showed that BCP and β-TCP could support macrophages attachment, proliferation, and spreading favorably, as well as promote gene expressions of inflammatory related cytokines (IL-1, IL-6, MCP-1, and TNF-α) and growth factors (TGF-β, FGF, PDGF, VEGF, IGF, and EGF). BCP showed a facilitating function on the gene expressions earlier than β-TCP. Further coculture experiments performed in vitro demonstrated that the CMs containing various increased cytokines for macrophages pre-culture could significantly promote MSCs osteogenic differentiation, which was confirmed by the gene expressions of osteogenic specific markers and the intracellular OCN product accumulation under the stimulation of BCP and β-TCP ceramics. Further evidence was found from the formation of mineralized nodules in BCM and TCM. In addition, this study showed a concise relationship between Ca-P ceramic induced inflammation and its osteoinductivity that the increased cytokines and growth factors from macrophages could promote MSCs osteogenic differentiation.

Role of serum allograft inflammatory factor-1 (AIF-1) in Egyptian type 2 diabetic patients

Background. Diabetes mellitus (DM) is a powerful and independent risk factor for cardiovascular disease. The atherosclerosis process in diabetes is indistinguishable from that of the nondiabetic population, but it begins earlier and is often more extensive and more severe. AIF-1 promotes chemotaxis, spreading and migration of macrophages and vascular smooth muscle cells (VSMCs) which suggest a role of AIF-1 in the atherosclerotic plaque formation. Thus, this study determines the role of AIF-1 in the Egyptian type 2 diabetic patients. Results. The level of AIF-1 was significantly higher in the type 2 diabetic group when compared to the control group (p = 0.000). In type 2 diabetic patients group, there was a significant positive correlation between CIMT and AIF-1 (r = 0.468, p = 0.000). In addition to the positive correlation between CIMT and AIF-1, CIMT in regression model analysis was significantly positive contributing to the outcome variable (AIF-1) (p < 0.05), denoting the possible role of elevated serum AIF-1 level in atherosclerotic process with further studies on larger scale needed.

Nanostructural Surfaces with Different Elastic Moduli Regulate the Immune Response by Stretching Macrophages.

A proper immune response is key for the successful implantation of biomaterials, and designing and fabricating biomaterials to regulate immune responses is the future trend. In this work, three different nanostructures were constructed on the surface of titanium using a hydrothermal method, and through a series of in vitro and in vivo experiments, we found that the aspect ratio of nanostructures can affect the elastic modulus of a material surface and further regulate immune cell behaviors. This work demonstrates that nanostructures with a higher aspect ratio can endow a material surface with a lower elastic modulus, which was confirmed by experiments and theoretical analyses. The deflection of nanostructures under the cell adsorption force is a substantial factor in stretching macrophages to enhance cell adhesion and spreading, further inducing macrophage polarization toward the M1 phenotype and leading to intense immune responses. In contrast, a nanostructure with a lower aspect ratio on a material surface leads to a higher surface elastic modulus, making deflection of the material difficult and creating a surface that is not conducive to macrophage adhesion and spreading, thus reducing the immune response. Moreover, molecular biology experiments indicated that regulation of the immune response by the elastic modulus is primarily related to the NF-κB signaling pathway. These findings suggest that the immune response can be regulated by constructing nanostructural surfaces with the proper elastic modulus through their influence on cell adhesion and spreading, which provides new insights into the surface design of biomaterials.

Association of CD47 with Integrin Mac-1 (αMβ2, CD11b/CD18) Regulates Macrophage Responses

CD47 is a cell surface receptor, which is expressed by virtually all cells in the body, including immune cells. CD47 has originally been identified as an integrin-associated protein (IAP) and shown to associate with several integrins that belong to the β1 and β3 subfamilies. In addition, association of CD47 with a member of the β2 subfamily, integrin αLβ2, has also been reported. In neutrophils, CD47 mediates a number of integrin αvβ3-dependent functions, including adhesion, migration and phagocytosis. Surprisingly, the association of CD47 with integrin αMβ2 (Mac-1, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, has not been documented. Furthermore, while the major focus of recent studies was the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 expressed on the surface of macrophages influences the responses of these cells has not been addressed. In the present study, we demonstrated that an association of CD47 with Mac-1 regulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. In addition, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional association of CD47 with Mac-1 implied that similar to other integrins, Mac-1 might form a complex with CD47. Indeed, co-immunoprecipitation experiments using peritoneal mouse macrophages, the IC21 murine macrophage cell line and Mac-1-expressing HEK293 cells revealed that Mac-1 forms a complex with CD47. The cis interaction between Mac-1 and CD47 was also detected using the proximity ligation assay. Together, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions. Studies to determine the structural requirements for the physical interaction between Mac-1 and CD47 are in progress. DisclosuresNo relevant conflicts of interest to declare.

Spatial confinement downsizes the inflammatory response of macrophages.

Macrophages respond to chemical/metabolic and physical stimuli, but their effects cannot be readily decoupled in vivo during pro-inflammatory activation. Here, we show that preventing macrophage spreading by spatial confinement, as imposed by micropatterning, microporous substrates or cell crowding, suppresses late lipopolysaccharide (LPS)-activated transcriptional programs (biomarkers IL-6, CXCL9, IL-1β, and iNOS) by mechanomodulating chromatin compaction and epigenetic alterations (HDAC3 levels and H3K36-dimethylation). Mechanistically, confinement reduces actin polymerization, thereby lowers the LPS-stimulated nuclear translocation of MRTF-A. This lowers the activity of the MRTF-A-SRF complex and subsequently downregulates the inflammatory response, as confirmed by chromatin immunoprecipitation coupled with quantitative PCR and RNA sequencing analysis. Confinement thus downregulates pro-inflammatory cytokine secretion and, well before any activation processes, the phagocytic potential of macrophages. Contrarily, early events, including activation of the LPS receptor TLR4, and downstream NF-κB and IRF3 signalling and hence the expression of early LPS-responsive genes were marginally affected by confinement. These findings have broad implications in the context of mechanobiology, inflammation and immunology, as well as in tissue engineering and regenerative medicine.

F2-isoprostanes affect macrophage migration and CSF-1 signalling

F2-isoprostanes (F2-IsoP) are formed in vivo via free radical peroxidation of arachidonic acid. Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and F2-IsoP have been detected in atherosclerotic plaque. Colony stimulating factor-1 (CSF-1) is essential to macrophage survival, proliferation and differentiation and has been detected in human atherosclerotic plaques. Accumulation of macrophages within the vascular wall is an important component of atherosclerosis but little is known about the effect of F2-IsoP on the migration of these cells. Our aim was to examine the effect of free and lipid-bound 15-F2t-isoprostane (15-F2t-IsoP) on macrophage migration and investigate the signalling pathways involved.Mouse macrophages (cell line BAC1.2F5) were pre-incubated with 15-F2t-IsoP (free, bound to cholesterol or monoacylglycerol or within oxidized phospholipid) and cell migration was assessed using chemotaxis towards CSF-1 in Boyden chambers. Migration was also measured using the wound healing assay with primary mouse bone marrow derived macrophages.We showed that 15-F2t-IsoP dose-dependently inhibited BAC1.2F5 macrophage spreading and adhesion but stimulated their migration towards CSF-1, with maximum effect at 10 µM. Analysis of CSF-1 stimulated signalling pathways in BAC1.2F5 macrophages showed that phosphorylation of Akt, a key mediator of cell migration, and one of its regulators, the mTORC2 component, Rictor, was significantly decreased. In contrast, phosphorylation of the adhesion kinases, FAK and Pyk2, and the adhesion scaffold protein, paxillin, was enhanced after treatment with 15-F2t-IsoP. Mouse bone marrow macrophages were transfected with FAK or Pyk2 small interfering RNA (siRNA) to examine the role of FAK and Pyk2 in 15-F2t-IsoP signalling. Pyk2 silencing inhibited 15-F2t-IsoP-induced reduction in cell area and phospho-paxillin adhesion numbers. The size distribution of adhesions in the presence of 15-F2t-IsoP was also affected by Pyk2 silencing and there was a trend for Pyk2 silencing to reduce 15-F2t-IsoP-stimulated macrophage migration.These results demonstrate that 15-F2t-IsoP affects macrophage adhesions and migration, which are integral components of macrophage involvement in atherosclerosis.

Ziprasidone, a second-generation antipsychotic drug, triggers a macrophage inflammatory response in vitro

Antipsychotic drugs are used to treat schizophrenia and other psychiatric disorders. However, most of these drugs present side effects causing obesity and other serious metabolic alterations that correlate with grade of chronic inflammation. In contrast, ziprasidone’s (ZIP) metabolic side effects are attenuated relative to those of other antipsychotic drugs, but some reports suggest that this drug could cause allergic, hypersensitive reactions in susceptible patients. At present, the mechanism of ZIP’s effect on peripheral inflammatory metabolism is not well characterized. We conducted an in vitro study to evaluate the effect of ZIP on a macrophage cell line (RAW 264.1). Our results showed that in non-activated macrophage cells, ZIP exposure initiated macrophage spreading; increased cellular proliferation, as evaluated by MTT and flow cytometry assays; and presented higher levels of oxidant molecules involved in the inflammatory response (nitric oxide, superoxide, reactive oxygen species), and proinflammatory cytokines (IL-1, IL-6, TNFα, INFγ). Levels of IL-10, an anti-inflammatory cytokine were lower in ZIP-exposed cells. These effects were less potent than those caused by the positive control for inflammation induction (phytohemagglutinin), and more intense than the effects of lithium (LI), which was used as an anti-inflammatory molecule. ZIP also modulated cytokine gene expression. Taken together, these data suggest that ZIP can produce a peripheral inflammatory response, and this response may explain the allergen-inflammatory response observed in some patients treated with this antipsychotic drug.

Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor

Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tissue localization. Their susceptibility to HIV-1 is subject to variations from permissiveness to resistance, owing in part to regulatory microRNAs. Here, we used RNA sequencing (RNA-seq) to examine the expression of >400 microRNAs in productively infected and bystander cells of HIV-1-exposed macrophage cultures. Two microRNAs upregulated in bystander macrophages, miR-221 and miR-222, were identified as negative regulators of CD4 expression and CD4-mediated HIV-1 entry. Both microRNAs were enhanced by tumor necrosis factor alpha (TNF-α), an inhibitor of CD4 expression. MiR-221/miR-222 inhibitors recovered HIV-1 entry in TNF-α-treated macrophages by enhancing CD4 expression and increased HIV-1 replication and spread inmacrophages by countering TNF-α-enhanced miR-221/miR-222 expression in bystander cells. In linewith these findings, HIV-1-resistant intestinal myeloid cells express higher levels of miR-221 than peripheral blood monocytes. Thus, miR-221/miR-222 act as effectors of the antiviral host response activated during macrophage infection that restrict HIV-1 entry.

A novel moonlight function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for immunomodulation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme, which generates NADH in glycolysis. Our previous study revealed a novel role of exogenous GAPDH in the amelioration of lipopolysaccharide (LPS)-induced sepsis-related, severe acute lung injury (ALI) in mice. Here, we show the effect of extracellular GAPDH on the physiological functions of macrophages, which play an important role in the onset of sepsis and ALI. GAPDH has no effect on cell viability, while it strongly suppressed cell adhesion, spreading, and phagocytic function of LPS-stimulated macrophages. GAPDH treatment significantly reduced tumor necrosis factor (TNF)-α, while it induced interleukin (IL)-10 production from LPS-stimulated macrophages in a dose-dependent manner. It is noteworthy that heat inactivation of GAPDH lost its immunomodulatory activity. Correspondingly, NADH significantly inhibited TNF-α and enhanced IL-10 production with elevation of both M1/M2 macrophage markers. These data suggest that extracellular GAPDH induces intermediate M1/M2 macrophages for termination of inflammation, partly through its enzyme activity for generation of NADH. © 2018 BioFactors, 44(6):597-608, 2018.

Oxidative stress and inflammation caused by n-Hexyl salicylate in mouse skin: the effectiveness of flavonoids

&lt;p&gt;Reactive oxygen species (ROS) play a role in a numbered of degenerative conditions including psoriasis. Psoriasis is a chronic inflammatory disease who’s the etiopathogenesis is not yet completely understood, and therefore there is no standardized therapeutical approach. Flavonoids, recognized as potent antioxidants, are multifunctional molecules that can act as anti-inflammatory and antiproliferative agents through the modulation of multiple signaling pathways. The present study was designed to investigate the protective role of flavonoids [quercetin, chrysin, curcumin or Epigallocatechin 3-gallate (EGCG)] against n-Hexyl salicylate (HXS)-induced oxidative stress and inflammation in skin. Anti-oxidative and anti-inflammatory effect of flavonoids is quantified by histopathological assessment of skin, measuring the levels of lipid peroxidation and glutathione (GSH) in the skin, total number of inflammatory cells in peritoneal cavity, macrophage spreading index, and hematological and biochemical parameters.&lt;/p&gt;&lt;p&gt;Topically applied of n-Hexyl salicylate caused significant increase in lipid peroxidation and decrease in GSH, which is accompanied by an increase&lt;strong&gt; &lt;/strong&gt;&lt;em&gt;total number of inflammatory cells in skin and peritoneal cavity, functional activity of macrophages, and enzymatic activity of ALP and AST.&lt;/em&gt; In contrast, topically applied 5 % preparation of flavonoids (quercetin, chrysin, curcumin or EGCG) with HXS effectively prevented these alterations and maintained the antioxidant status.&lt;/p&gt;&lt;p&gt;The results suggest that flavonoid preparations can serve as a potent antioxidant and anti-inflammatory agents in psoriatic-like skin lesions, without toxic effects.&lt;/p&gt;

Honey and quercetin reduce ochratoxin A-induced DNA damage in the liver and the kidney through the modulation of intestinal microflora

ABSTRACTThis study was conducted to evaluate the interactions between the gut microbiota, ochratoxin A and functional food such as honey and quercetin, and the consequences of these interactions on ochratoxin-induced DNA damage in blood, liver and kidney cells. Honey (2 g kg−1) or Quercetin (50 mg kg−1) was applied to mice by intragastric application every day for 15 days, immediately before ochratoxin treatment (100 µg kg−1). We investigated colonic probiotic bacteria count, β-glucuronidase activity, the alkaline comet assay in blood, liver and kidney, the number of cells in the peritoneal cavity, macrophage spreading index and hematological and biochemical parameters. Honey and QU may reduce ochratoxin-induced DNA damage in the liver and kidney, β-glucuronidase activity and inflammation, partly through increasing the colon Bifidobacteria and Lactobacilli counts. The obtained results suggest that honey and QU counteracted the OTA-induced toxicity due to their bifidogenic activity and antigenotoxic activity.

Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages.

Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec(-) T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1.

Drosophila WASH is required for integrin-mediated cell adhesion, cell motility and lysosomal neutralization.

The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH; also known as Washout in flies) is a conserved actin-nucleation-promoting factor controlling Arp2/3 complex activity in endosomal sorting and recycling. Previous studies have identified WASH as an essential regulator in Drosophila development. Here, we show that homozygous wash mutant flies are viable and fertile. We demonstrate that Drosophila WASH has conserved functions in integrin receptor recycling and lysosome neutralization. WASH generates actin patches on endosomes and lysosomes, thereby mediating both aforementioned functions. Consistently, loss of WASH function results in cell spreading and cell migration defects of macrophages, and an increased lysosomal acidification that affects efficient phagocytic and autophagic clearance. WASH physically interacts with the vacuolar (V)-ATPase subunit Vha55 that is crucial to establish and maintain lysosome acidification. As a consequence, starved flies that lack WASH function show a dramatic increase in acidic autolysosomes, causing a reduced lifespan. Thus, our data highlight a conserved role for WASH in the endocytic sorting and recycling of membrane proteins, such as integrins and the V-ATPase, that increase the likelihood of survival under nutrient deprivation.

Covalent immobilization of glycosaminoglycans to reduce the inflammatory effects of biomaterials.

The inflammatory responses evoked by artificial organs and implantation of devices like biosensors and guide wires can lead to acute and chronic inflammation, largely limiting the functionality and longevity of the devices with negative effects on patients. The present study aimed to reduce the inflammatory responses to biomaterials by covalent immobilization of glycosaminoglycans (GAGs) on amino-terminated surfaces used as model biomaterials here. Water contact angle (WCA) and zeta potential measurements showed a significant increase in wettability and negative charges on the GAG-modified surfaces, respectively, confirming the successful immobilization of GAGs on the amino-terminated surfaces. THP-1-derived macrophages were used as a model cell type to investigate the efficacy of GAG-modified surfaces in modulating inflammatory responses. It was found that macrophage adhesion, macrophage spreading morphology, foreign body giant cell (FBGC) formation, as well as β1 integrin expression and interleukin-1β (IL-1β) production were all significantly decreased on GAG-modified surfaces compared to the initial amino-terminated surface. This study demonstrates the potential of covalent GAG immobilization to reduce the inflammatory potential of biomaterials in different clinical settings.