Macrophage Fusion Research Articles

An in vitro model of foreign body giant cell formation mediated by damage-associated molecular patterns

Event Abstract Back to Event An in vitro model of foreign body giant cell formation mediated by damage-associated molecular patterns Jeremy Antonyshyn1, 2 and Lindsay E. Fitzpatrick1 1 Queen's University, Department of Chemical Engineering, Canada 2 University of Toronto, Institute of Biomaterials & Biomedical Engineering, Canada Introduction: Protein adsorption to biomaterials is a well-known phenomenon thought to contribute to the progression of the foreign body reaction (FBR). While the contribution of serum proteins has been studied extensively, those derived from tissues damaged during implantation have received little attention. Toll-like receptors (TLRs) may mount an inflammatory response against these damage-associated molecular patterns (DAMPs) through their activation of NF-κB and AP-1 transcription factors[1]. The purpose of this study was to determine whether TLR-mediated macrophage recognition of DAMPs adsorbed to biomaterial surfaces contributes to the progression of the FBR. Methods: Fetal bovine serum (FBS) and 3T3 fibroblast lysate were used as sources of serum proteins and DAMPs, respectively. Total protein concentrations were determined by microBCA assay. Tissue culture polystyrene (TCPS) was incubated in lysate and/or FBS, then rinsed with phosphate-buffered saline (PBS). Activation of NF-κB/AP-1 in RAW-Blue macrophages induces expression of secreted embryonic alkaline phosphatase (SEAP), the enzymatic activity of which can be quantified with the QUANTI-Blue assay. These cells, cultured in 10% heat-inactivated FBS, were exposed to TCPS alone (negative control), lipopolysaccharide (LPS; positive control), and lysate/FBS-adsorbed TCPS. Culture was ended after 24hrs. Morphology of May-Grünwald-Giemsa –stained cells was evaluated. IL-4 and IFN-γ concentrations were determined by ELISAs. NF-κB/AP-1 –dependent SEAP activity was quantified. All experiments were run in triplicate, ANOVAs with Tukey’s post-hoc analyses used, and p<0.05 accepted as statistically significant. All values reported as mean±SD. Results and Discussion: Lysate induced significantly greater macrophage fusion than TCPS (39.2±13.9% vs 0.5±0.3%, respectively; p<0.05), yielding multinucleated cells (27.0±19.9 nuclei/cell; 9068.0±6548.0µm2) of irregular morphology and random nuclear arrangement (Fig 1). Previous models have relied on IL-4 and IFN-γ to induce fusion[2], but neither was detected suggesting an alternative mechanism. Lysate induced NF-κB/AP-1 activation to the same extent as known TLR-agonist LPS (11.4±0.1 vs 11.9±1.1 fold greater than TCPS, respectively), which was significantly greater than that induced by FBS (0.3±0.1 fold; p<0.001). While this suggests that TLRs mount an inflammatory response against DAMPs but not serum proteins, both compete for adsorption as evidenced by the dose-dependent suppression of NF-κB/AP-1 activity with dilution of lysate in FBS but not PBS (p<0.0001; Fig 2). Importantly, lysate continued to elicit significant activation compared to FBS when comprising as little as 0.06% of total protein added (p<0.05), supporting the relevance of DAMPs to the FBR in vivo. Conclusion: These findings indicate that DAMPs-adsorbed biomaterials elicit an inflammatory response in macrophages and promote their fusion to yield foreign body giant cells, the hallmark of the FBR. Canada Foundation for Innovation’s (CFI) John R. Evans Leaders Fund (JELF); Queen’s University Senate Advisory Research Committee (SARC) Grant

Serine protease inhibition attenuates foreign body giant cell formation

Event Abstract Back to Event Serine protease inhibition attenuates foreign body giant cell formation Aisling N. O'Carroll1, Brendan F. Gilmore1, Colin P. Mccoy1 and Louise Carson1 1 Queen's University Belfast, School of Pharmacy, United Kingdom Introduction: Upon biomaterial implantation, macrophages (MΦ) adhere to the material surface and can fuse to form large, multinucleated Foreign Body Giant Cells (FBGCs) [1]. MΦ adherence and FBGC formation on implanted biomaterial surfaces are pivotal events in the progression of a Foreign Body Response (FBR). The host response is further compounded by protease release from FBGCs, during a process described as ‘frustrated phagocytosis’, causing both biomaterial and tissue component degradation in the peri-implant region [2]. Previous studies highlight roles for matrix metalloproteases (MMP)-9 in MΦ fusion [3]. However, little is known regarding the role of serine proteases in FBGC biology. Work presented details the proteolytic activity of FBGCs and effect of serine protease inhibition on the fusogenic capabilities of primary murine MΦs. Materials and Methods: Fusion assays were performed using primary murine bone marrow derived macrophages (BMM) isolated from WT C57BL/6 mice, and treated with IL-4 (10 ng/ml), GM-CSF (10 ng/ml), and 100 µM affinity-based biotinylated inhibitor probes (or vehicle control) targeting three classes of serine proteases; trypsin-like, chymotrypsin-like and elastase-like proteases [4]. Each inhibitor probe was tested alone, and in combination to highlight possible synergistic activity. Protease temporal expression and activity during FBGC formation was analysed by SDS-PAGE, Zymography and Western Blotting from cell-conditioned media and whole cell lysates. Results: Increased serine protease secretion in FBGCs at Day 2, 5 and 7 is observed, when compared with non-fused mononuclear controls. These proteases range from 38-49 kDa in size. FBGC formation is reduced by almost 50% when subject to protease inhibitor treatment. Statistical analyses show BMM treatment with inhibitors directed towards trypsin-like (P < 0.01), chymotrypsin-like (P < 0.001) and elastase-like (P < 0.05) activity results in a significant decrease in percentage fusion when compared with controls. Combined treatment with all three protease inhibitors dramatically reduces FBGC formation in vitro. Discussion: Western blotting, ultilizing the biotinylated inhibitor probes, suggests an increase in protease activity in fused BMMs overtime when compared with unfused controls, in particular an increase in trypsin-like and chymotrypsin-like actiivity at Day 5 and 7. Simultaneous treatment with all three serine inhibitors targeting chymotrypsin-like, trypsin-like and elastase-like serine proteases results in a significant reduction in FBGC formation, over and above each inhibitor alone, and is indicative of a synergistic effect. Such treatment represents a viable novel strategy to abrogate macrophage fusion. Conclusion: Together, these results denote a function for serine proteases in MΦ fusion and FBGC biology. Serine proteases may represent potential novel therapeutic targets in efforts to prevent the FBR – be it due to their influential role initiating intracellular signalling or indeed orchestrating cytoskeletal reorganisation during the fusion process. Future work will centre on the identification and functional characterisation of these serine proteases, unmasking their mechanistic role in FBGC formation.

Multinucleated Giant Cells Are Specialized for Complement-Mediated Phagocytosis and Large Target Destruction.

SummaryMultinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.

Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

Macrophage-tumor cell fusions from peripheral blood of melanoma patients.

BackgroundWhile the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.MethodsWe isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.FindingsMorphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.Conclusions and HypothesisApparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.

E-cadherin expression in macrophages dampens their inflammatory responsiveness in vitro, but does not modulate M2-regulated pathologies in vivo.

IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested.

Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells.

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast‑associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied.

Nanochannels formed on TiZr alloy improve biological response

In the present work we report the fabrication of non-thickness-limited 1D nanostructures with nanochannelar structure by anodization of Ti50Zr alloy in hot glycerol-phosphate electrolyte. These nanochannelar structures show high and adjustable aspect ratios and provide as-formed already partial crystallinity for nanochannels. In vitro studies were performed to assess the inflammatory response to nanochannel coated surfaces using RAW 264.7 macrophages. The results show that these nanochannels yield a reduced amount of metabolically-active macrophages, low potential to induce macrophage fusion into FBGC, and low concentration of pro-inflammatory cytokines in the culture medium. Moreover, higher hydrophilicity and lower corrosion rates were registered, compared to compact oxide. Collectively, the results indicate a more favorable cellular response on such nanoscale topography as compared to compact oxide control substrate, and suggest that surface architecture design of nanochannel type on implant materials holds promise for biomedical applications. Statement of SignificanceThe use of titanium and its alloys in biomedical devices has been extensively investigated, especially for alloys possessing inherent antibacterial properties such as TiZr alloys. We report for the first time the growth of mesoporous structures, aligned oxide nanochannels, on Ti50Zr alloy. The advantages of these nanochannelar surfaces are a high surface area, a long range ordered nanoscale topology and already partial crystallinity in the as-grown state. In vitro studies performed on RAW 264.7 macrophages demonstrate the potential of nanochannels to lower the inflammatory response, thus reducing the foreign body reaction against Ti50Zr biomedical implants and promoting the successful integration of the implant.

The effects of red ginseng saponin fraction-A (RGSF-A) on phagocytosis and intracellular signaling in Brucella abortus infected RAW 264.7 cells

This study indicated that RGSF-A caused a marked reduction in the adherence, internalization and intracellular growth of Brucella abortus in RGSF-A-treated cells. Furthermore, a decline in the intensity of F-actin fluorescence was observed in RGSF-A-treated cells compared with untreated B. abortus-infected cells. In addition, an evaluation of phagocytic signaling proteins by Western blot analysis revealed an apparent reduction of ERK and p38α phosphorylation levels in B. abortus-infected RGSF-A-treated cells compared with the control. Upon intracellular trafficking of the pathogen, a higher number of B. abortus-containing phagosomes colocalized with LAMP-1 in RGSF-A-treated cells compared with control cells. These results strongly suggest that inhibition of B. abortus uptake could be mediated by suppression in the activation of MAPKs signaling proteins phospho-ERK 1/2, and p38 levels. On the other hand, inhibition of intracellular replication results from the enhancement of phagolysosome fusion in host macrophages. This study highlights the phagocytic and intracellular modulating effect of RGSF-A and its potential as an alternative remedy to control B. abortus infection.

Foreign body granulomas after the use of dermal fillers: pathophysiology, clinical appearance, histologic features, and treatment.

A foreign body granuloma is a non-allergic chronic inflammatory reaction that is mainly composed of multinucleated giant cells. Foreign body granulomas may occur after the administration of any dermal filler. Factors such as the volume of the injection, impurities present in the fillers, and the physical properties of fillers affect granuloma formation. The formation of granulomas involves five phases: protein adsorption, macrophage adhesion, macrophage fusion, and crosstalk. The clinical and pathologic features of granulomas vary depending on the type of filler that causes them. Foreign body granulomas can be treated effectively with intralesional corticosteroid injections. Surgical excisions of granulomas tend to be incomplete because granulomas have ill-defined borders and moreover, surgical excisions may leave scars and deformities.

Alteration of Lysosome Fusion and Low-grade Inflammation Mediated by Super-low-dose Endotoxin

Subclinical super-low-dose endotoxin LPS is a risk factor for the establishment of low-grade inflammation during the pathogenesis and progression of chronic diseases. However, the underlying mechanisms are not well understood. At the cellular level, a disruption of lysosome fusion with endosomes or autophagosomes may contribute to the potentiation of low-grade inflammation. In this study, we identified that subclinical super-low-dose endotoxin LPS can potently inhibit the process of endosome acidification and lysosome fusion with endosomes or autophagosomes in primary macrophages. Super-low-dose LPS induced the inhibitory phosphorylation of VPS34, thus leading to the disruption of endosome-lysosome fusion. This effect may depend upon the clearance and relocation of Tollip in macrophages by super-low-dose LPS. Consistent with this notion, Tollip-deficient macrophages had constitutively elevated levels of VPS34 inhibitory phosphorylation and constitutive disruption of endosome-lysosome fusion. By employing a skin excision wound-healing model, we observed that Tollip-deficient mice had significantly elevated levels of cell stress and reduced wound repair. This study reveals a novel mechanism responsible for the modulation of endosome-lysosome fusion and low-grade inflammation in innate macrophages.

The unappreciated intracellular lifestyle of Blastomyces dermatitidis.

Blastomyces dermatitidis, a dimorphic fungus and the causative agent of blastomycosis, is widely considered an extracellular pathogen, with little evidence for a facultative intracellular lifestyle. We infected mice with spores, that is, the infectious particle, via the pulmonary route and studied intracellular residence, transition to pathogenic yeast, and replication inside lung cells. Nearly 80% of spores were inside cells at 24 h postinfection with 10(4) spores. Most spores were located inside of alveolar macrophages, with smaller numbers in neutrophils and dendritic cells. Real-time imaging showed rapid uptake of spores into alveolar macrophages, conversion to yeast, and intracellular multiplication during in vitro coculture. The finding of multiple yeast in a macrophage was chiefly due to intracellular replication rather than multiple phagocytic events or fusion of macrophages. Depletion of alveolar macrophages curtailed infection in mice infected with spores and led to a 26-fold reduction in lung CFU by 6 d postinfection versus nondepleted mice. Phase transition of the spores to yeast was delayed in these depleted mice over a time frame that correlated with reduced lung CFU. Spores cultured in vitro converted to yeast faster in the presence of macrophages than in medium alone. Thus, although advanced B. dermatitidis infection may exhibit extracellular residence in tissue, early lung infection with infectious spores reveals its unappreciated facultative intracellular lifestyle.

New therapeutic perspectives – amyloid removal

Background In systemic amyloidosis, disease is caused by extracellular accumulation of amyloid fibrils which, unlike other interstitial debris, are not cleared and which disrupt tissue structure and function. Direct removal of amyloid deposits is required to preserve and possibly restore tissue and organ function. We are targeting serum amyloid P component (SAP) for this purpose. SAP normal plasma protein is a normal plasma protein which binds to all amyloid fibrils and is thus always present in all human amyloid deposits. Administration of hexanoyl bis (D–proline) (CPHPC) swiftly depletes circulating SAP but leaves some SAP in amyloid deposits as an amyloidspecific antigen target. In human SAP transgenic mice with systemic AA amyloidosis, CPHPC treatment, to deplete human SAP from the plasma, followed by a single dose of anti-human SAP antibodies produced swift, almost complete, clearance of visceral amyloid (Bodin et al. Nature, 2010;468:93-7). In a mouse SAA transgenic systemic amyloidosis model, which uniquely includes cardiac amyloid, a second dose of anti–SAP antibody, after the first dose had eliminated massive liver and spleen deposits, significantly removed amyloid from the heart (Simons et al. Proc Natl Acad Sci USA. 2013;110:1611520). Amyloid clearance by anti-SAP antibody required classical complement pathway activation and macrophages. Amyloid destruction was mediated by multinucleated giant cells (MGCs), formed by macrophage fusion. MGCs have abundant surface membrane ruffles, enabling the engulfment of very large complement opsonised amyloid targets which were then swiftly destroyed within phagolysosomes. Amyloid clearance was maximal by 14 days. No ill effects were detected. After licensing this new treatment in February 2009, GlaxoSmithKline (GSK) fully humanised our optimal mouse monoclonal anti-SAP antibody and prepared for the first in human clinical study that started in June 2013. We have lately reported that a single dose of humanized monoclonal anti-SAP antibody, following depletion of circulating SAP by CPHPC, substantially reduced the amyloid load, especially from the liver, in patients with systemic AL, AA and AApoAI amyloidosis, (Richards et al, New Engl J Med, July 15 2015; DOI: 10.1056/NEJMoa1504942).

Foreign Body Reaction to Subcutaneous Implants.

Subcutaneously implanted materials trigger the host's innate immune system, resulting in the foreign body reaction. This reaction consists of protein adsorption on the implant surface, inflammatory cell infiltration, macrophage fusion into foreign body giant cells, fibroblast activation and ultimately fibrous encapsulation. This series of events may affect the function of subcutaneous implants, such as inhibition of drug diffusion from long-acting drug delivery depots and medical device failure. The foreign body reaction is a complex phenomenon and is not yet fully understood; ongoing research studies aim to elucidate the cellular and molecular dynamics involved. Recent studies have revealed information about the specific role of macrophages and their differential activation towards pro- and anti-inflammatory states, as well as species differences in the timing of collagen deposition and fibrosis. Understanding of the diverse processes involved in the foreign body reaction has led to multiple approaches towards its negation. Delivery of tissue response modifiers, such as corticosteroids, NSAIDs, antifibrotic agents, and siRNAs, has been used to prevent or minimize fibrosis. Of these, delivery of dexamethasone throughout the implantation period is the most common method to prevent inflammation and fibrosis. More recent approaches employ surface modifications to minimize protein adsorption to 'ultra-low' levels and reduce fibrosis. However, the diverse nature of the processes involved in the foreign body reaction favor the use of corticosteroids due to their wide spectrum action compared to other approaches. To date, combination approaches, such as hydrophilic coatings that reduce protein adsorption combined with delivery of dexamethasone are the most effective.

Late stages of the synchronized macrophage fusion in osteoclast formation depend on dynamin.

Macrophage fusion that leads to osteoclast formation is one of the most important examples of cell-cell fusion in development, tissue homoeostasis and immune response. Protein machinery that fuses macrophages remains to be identified. In the present study, we explored the fusion stage of osteoclast formation for RAW macrophage-like murine cells and for macrophages derived from human monocytes. To uncouple fusion from the preceding differentiation processes, we accumulated fusion-committed cells in the presence of LPC (lysophosphatidylcholine) that reversibly blocks membrane merger. After 16h, we removed LPC and observed cell fusion events that would normally develop within 16h develop instead within 30-90min. Thus, whereas osteoclastogenesis, generally, takes several days, our approach allowed us to focus on an hour in which we observe robust fusion between the cells. Complementing syncytium formation assay with a novel membrane merger assay let us study the synchronized fusion events downstream of a local merger between two plasma membranes, but before expansion of nascent membrane connections and complete unification of the cells. We found that the expansion of membrane connections detected as a growth of multinucleated osteoclasts depends on dynamin activity. In contrast, a merger between the plasma membranes of the two cells was not affected by inhibitors of dynamin GTPase. Thus dynamin that was recently found to control late stages of myoblast fusion also controls late stages of macrophage fusion, revealing an intriguing conserved mechanistic motif shared by diverse cell-cell fusion processes.

Abstract 3959: Depleting macrophage or blocking cell fusion machinery ameliorates intestine fibrosis after radiation

Abstract Purpose Delayed radiation enteritis is a frequent side effect of abdominal radiotherapy. Recent studies reveal bone marrow (BM) cells repopulate damaged tissue and contribute to repair in non-hematopoietic tissues. Fusion between BM and somatic cells is enhanced by chronic inflammation. Furthermore, BM cells (BMC) can trigger fibrotic response in damaged organ. The role of cell fusion in the development of chronic intestine fibrosis after radiotherapy needs further investigation. Materials and Methods To evaluate cell fusion and fibrosis, gender-mismatched BM transplantation (BMT) were used. Immunoblots of macrophage fusion receptor (MFR), CD47 and fibronectin, connective tissue growth factor (CCN2) were measured. BMT with CD11b (+), CD3 (+) or B220 (+) enriched BMC were applied. The mice were treated with macrophage depleting agents, clodronate liposome, after radiotherapy and BMT. In vitro co-culture study of human intestine stromal and mouse BMC was established. Furthermore, we used Rac 1 inhibitor, MMP9 neutralizing antibody and siRNA of MFR within co-culture system and animal model. Results Using triple stains of Y chromosome fluorescence in situ hybridization, GFP and hematopoietic cell markers including F4/80, CD3, B220, we found BM derived macrophages contributed majorly to cell fusion (63.4%, 47.5% and 9.6%, respectively, p=0.033) within intestine of mice after radiotherapy and BMT. The fusion within intestine decreased 57% after treatment with clodronate liposome. BMT from CD11b(+), CD3(+) or B220(+) enriched donor BMC also revealed prominent fusion only in mice receiving CD11b(+) one. The level of intestine fibrosis was decreased by clodronate liposome or BMT of CD11b(-) BMC. In vitro study showed the rate of fusion decreased from 12.8% to 6.8% between intestine stromal cells with CD11b (-) compared to that with whole mouse BMCs. Fusion rate could be increased by increasing CD11b(+) BMCs (3.5% to 19.7%). Using Rac1 inhibitor, MMP9 antibody or MFR siRNA, the fusion rate decreased (19.7% to 4.6%; 11.8% to 5.6%; 31% to 19.6%, respectively). The expression of fibronectin, CCN2, MFR and CD47 paralleled the findings of fusion. Using MMP9 neutralizing antibody in mice receiving radiotherapy and BMT, we found decreased cell fusion and fibrosis within intestine. Conclusions BM derived CD11b(+) myelomonocytic cells or macrophage contribute majorly to cell fusion and fibrosis within intestine of mice after radiotherapy Depleting macrophage or targeting cell fusion machinery of macrophage suppresses intestine fibrosis after radiotherapy. Our study impacts not only for developing novel therapies to prevent radiation enteritis but also to avoid fibrosis after BM cell therapy in organ dysfunction. Citation Format: Ya-Hui Chang, Hui-Ju Ch'ang, Li-Mei Lin. Depleting macrophage or blocking cell fusion machinery ameliorates intestine fibrosis after radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3959. doi:10.1158/1538-7445.AM2014-3959

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway.

Phenotypic expression in human monocyte-derived interleukin-4-induced foreign body giant cells and macrophages in vitro: dependence on material surface properties.

The effects of different material surfaces on phenotypic expression in macrophages and foreign body giant cells (FBGC) were addressed using our in vitro system of interleukin (IL)-4-induced macrophage fusion and FBGC formation. Arginine-glycine-aspartate (RGD)-, vitronectin (VN)-, and chitosan (CH)-adsorbed cell culture polystyrene, carboxylated (C, negatively charged) polystyrene, and unmodified (PS, non-cell culture treated) polystyrene were compared for their abilities to support monocyte/macrophage adhesion and IL-4-induced macrophage fusion. Pooled whole cell lysates from four different donors were evaluated by immunoblotting for expression of selected components in monocytes, macrophages, and FBGC. In addition to RGD and VN as previously shown, we find that CH supports macrophage adhesion and FBGC formation, whereas C or PS support macrophage adhesion but do not permit macrophage fusion under otherwise identical conditions of IL-4 stimulation. Likewise, components related to macrophage fusion (CD206, CD98, CD147, CD13) are strongly expressed on RGD-, VN-, and CH-adsorbed surfaces but are greatly diminished or not detected on C or PS. Importantly, material surfaces also influence the FBGC phenotype itself, as demonstrated by strong differences in patterns of expression of HLA-DR, B7-2, B7-H1, and toll-like receptor (TLR)-2 on RGD, VN, and CH despite morphologic similarities between FBGC on these surfaces. Likewise, we observe differences in the expression of B7-2, α2-macroglobulin, TLR-2, and fascin-1 between mononuclear macrophages on C and PS. Collectively, these findings reveal the extent to which material surface chemistry influences macrophage/FBGC phenotype beyond evident morphological similarities or differences and identify CH as an FBGC-supportive substrate.

Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression.

Suppressed Expression of Homotypic Multinucleation, Extracellular Domains of CD172α (SIRP‐α) and CD47 (IAP) Receptors in TAMs UpRegulated by Hsp70–Peptide Complex in Dalton's Lymphoma

CD172α and CD47 are members of glycoprotein expressed on macrophages and various immune cells, promote immune recognition and T cell stimulation that priming phagocytosis of pathogens and apoptotic bodies and malignant cell. Tumour-releasing immunosuppressive factor promotes tumour growth and transforms the tumour resident M1 phenotype of macrophage to M2 phenotype (TAMs) that promotes tumour progression by downregulating the expression of different surface receptor including CD172α and CD47. Recent studies have reported that CD172α and CD47 are involved in the pathogenesis and promote malignancies such as lymphoma, leukaemia, melanoma, lung cancer and multiple myeloma, and their expression varies during infection and malignancies. Autologous Hsp70 is well recognized for its role in activating macrophages leading to enhance production of inflammatory cytokines. It has been observed that Hsp70 derived from normal tissues do not elicit tumour immunity, while Hsp70 preparation from tumour cell was able to elicit tumour immunity. However, the role of exogenous autologous hsp70 on the formation of giant cells is completely unknown. Therefore, in the present study, we sought to investigate the effect of Hsp70-peptide complex on the expression of CD172α and CD47 receptors in normal peritoneal macrophages (NMO) and TAMs. Finding shows that the expression of CD172α and CD47 enhances in TAMs and it reverts back the suppressed function of TAMs into M1 state of immunoregulatory phenotype that promotes tumour regression by enhanced multinucleation and phagocytosis of malignant cells and significantly enhances the homotypic fusion of macrophages and polykaryon formation in vitro by enhancing the expression of SIRPα and IAP.