TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αß and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

Generation of gene-of-interest knockouts in murine organoids using CRISPR-Cas9.

Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene's effects on many biological and pathological processes. Here, we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes, organoid cell sorting, and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis.

Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages.

Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, including macrophages and other myeloid cells. Here, we benchmark Myc inactivation in tumor cells against the contribution of its expression in myeloid cells of murine hosts that harbor endogenous or allograft tumors. Surprisingly, we observe that LysMCre-mediated Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade. Likewise, we find no evidence for Myc ablation to revert or antagonize the polarization of alternatively activated immunosuppressive macrophages. Thus, we surmise that systemic targeting of Myc activity may confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than reinvigorating the anti-tumor activity of macrophages.

Inhibition of HCK in myeloid cells restricts pancreatic tumor growth and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a low 5-year survival rate and is associated with poor response to therapy. Elevated expression of the myeloid-specific hematopoietic cell kinase (HCK) is observed in PDAC and correlates with reduced patient survival. To determine whether aberrant HCK signaling in myeloid cells is involved in PDAC growth and metastasis, we established orthotopic and intrasplenic PDAC tumors in wild-type and HCK knockout mice. Genetic ablation of HCK impaired PDAC growth and metastasis by inducing an immune-stimulatory endotype in myeloid cells, which in turn reduced the desmoplastic microenvironment and enhanced cytotoxic effector cell infiltration. Consequently, genetic ablation or therapeutic inhibition of HCK minimized metastatic spread, enhanced the efficacy of chemotherapy, and overcame resistance to anti-PD1, anti-CTLA4, or stimulatory anti-CD40 immunotherapy. Our results provide strong rationale for HCK to be developed as a therapeutic target to improve the response of PDAC to chemo- and immunotherapy.

IL-33-mediated mast cell activation promotes gastric cancer through macrophage mobilization.

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer.

Interleukin 33 Signaling Restrains Sporadic Colon Cancer in an Interferon-γ-Dependent Manner.

Interleukin 33 (IL33) is an inflammatory cytokine released during necrotic cell death. The epithelium and stroma of the intestine express large amounts of IL33 and its receptor St2. IL33 is therefore continuously released during homeostatic turnover of the intestinal mucosa. Although IL33 can prevent colon cancer associated with inflammatory colitis, the contribution of IL33 signaling to sporadic colon cancer remains unknown. Here, we utilized a mouse model of sporadic colon cancer to investigate the contribution of IL33 signaling to tumorigenesis in the absence of preexisting inflammation. We demonstrated that genetic ablation of St2 enhanced colon tumor development. Conversely, administration of recombinant IL33 reduced growth of colon cancer cell allografts. In reciprocal bone marrow chimeras, the concurrent loss of IL33 signaling within radioresistant nonhematopoietic, and the radiosensitive hematopoietic, compartments was associated with increased tumor burden. We detected St2 expression within the radioresistant mesenchymal cell compartment of the colon whose stimulation with IL33 induced expression of bona fide NF-κB target genes. Mechanistically, we discovered that St2 deficiency within the nonhematopoietic compartment coincided with increased abundance of regulatory T cells and suppression of an IFNγ gene expression signature, whereas IL33 administration triggered IFNγ expression by tumor allograft-infiltrating T cells. The decrease of this IFNγ gene expression signature was associated with more aggressive disease in human colon cancer patients, suggesting that lack of IL33 signaling impaired the generation of a potent IFNγ-mediated antitumor immune response. Collectively, our data reveal that IL33 functions as a tumor suppressor in sporadic colon cancer. Cancer Immunol Res; 6(4); 409-21. ©2018 AACR.

Inhibition of CD40-TRAF6 interactions by the small molecule inhibitor 6877002 reduces neuroinflammation.

BACKGROUND: The influx of leukocytes into the central nervous system (CNS) is a key hallmark of the chronic neuro-inflammatory disease multiple sclerosis (MS). Strategies that aim to inhibit leukocyte migration across the blood-brain barrier (BBB) are therefore regarded as promising therapeutic approaches to combat MS. As the CD40L-CD40 dyad signals via TNF receptor-associated factor 6 (TRAF6) in myeloid cells to induce inflammation and leukocyte trafficking, we explored the hypothesis that specific inhibition of CD40-TRAF6 interactions can ameliorate neuro-inflammation. METHODS: Human monocytes were treated with a small molecule inhibitor (SMI) of CD40-TRAF6 interactions (6877002), and migration capacity across human brain endothelial cells was measured. To test the therapeutic potential of the CD40-TRAF6-blocking SMI under neuro-inflammatory conditions in vivo, Lewis rats and C57BL/6J mice were subjected to acute experimental autoimmune encephalomyelitis (EAE) and treated with SMI 6877002 for 6 days (rats) or 3 weeks (mice). RESULTS: We here show that a SMI of CD40-TRAF6 interactions (6877002) strongly and dose-dependently reduces trans-endothelial migration of human monocytes. Moreover, upon SMI treatment, monocytes displayed a decreased production of ROS, tumor necrosis factor (TNF), and interleukin (IL)-6, whereas the production of the anti-inflammatory cytokine IL-10 was increased. Disease severity of EAE was reduced upon SMI treatment in rats, but not in mice. However, a significant reduction in monocyte-derived macrophages, but not in T cells, that had infiltrated the CNS was eminent in both models. CONCLUSIONS: Together, our results indicate that SMI-mediated inhibition of the CD40-TRAF6 pathway skews human monocytes towards anti-inflammatory cells with reduced trans-endothelial migration capacity, and is able to reduce CNS-infiltrated monocyte-derived macrophages during neuro-inflammation, but minimally ameliorates EAE disease severity. We therefore conclude that SMI-mediated inhibition of the CD40-TRAF6 pathway may represent a beneficial treatment strategy to reduce monocyte recruitment and macrophage activation in the CNS and has the potential to be used as a co-treatment to combat MS.

Generation of an inducible mouse model to reversibly silence Stat3.

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that has many essential roles during inflammation, development and cancer. Stat3 is therefore an attractive therapeutic target in many diseases. While current Stat3 knockout mouse models led to a better understanding of the role of Stat3, the irreversible nature of Stat3 ablation does not model the effects of transient Stat3 therapeutic inhibition, and does not inform on potential dosage effects of Stat3. Using RNAi technology, we have generated a new mouse model allowing the inducible and reversible silencing of Stat3 in vivo, which mirrors the effects of specific Stat3 therapeutic interference. We showed that upon Doxycycline-mediated activation of the Stat3 short-hairpin RNA, Stat3 expression was efficiently reduced by about 80% in multiple organs and cell types. Moreover, Stat3 reduction was sufficient to reduce tumor burden in a clinically-validated mouse model of gastric cancer. Finally, we demonstrated that Stat3 silencing during embryonic development led to reduced birth rate without leading to complete embryonic lethality, in contrast to full Stat3 ablation. In conclusion, this new mouse model will be invaluable to understand the effects of Stat3 therapeutic interference and Stat3 dosage effects.

The CD40-CD40L Dyad in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis.

The CD40-CD40L dyad is an immune checkpoint regulator that promotes both innate and adaptive immune responses and has therefore an essential role in the development of inflammatory diseases, including multiple sclerosis (MS). In MS, CD40 and CD40L are expressed on immune cells present in blood and lymphoid organs, affected resident central nervous system (CNS) cells, and inflammatory cells that have infiltrated the CNS. CD40-CD40L interactions fuel the inflammatory response underlying MS, and both genetic deficiency and antibody-mediated inhibition of the CD40-CD40L dyad reduce disease severity in experimental autoimmune encephalomyelitis (EAE). Both proteins are therefore attractive therapeutic candidates to modulate aberrant inflammatory responses in MS. Here, we discuss the genetic, experimental and clinical studies on the role of CD40 and CD40L interactions in EAE and MS and we explore novel approaches to therapeutically target this dyad to combat neuroinflammatory diseases.

Treatment with Trichuris suis soluble products during monocyte-to-macrophage differentiation reduces inflammatory responses through epigenetic remodeling.

Helminths have strong immunoregulatory properties that may be exploited in treatment of chronic immune disorders, such as multiple sclerosis and inflammatory bowel disease. Essential players in the pathogenesis of these diseases are proinflammatory macrophages. We present evidence that helminths modulate the function and phenotype of these innate immune cells. We found that soluble products derived from the Trichuris suis (TsSP) significantly affect the differentiation of monocytes into macrophages and their subsequent polarization. TsSPs reduce the expression and production of inflammatory cytokines, including IL-6 and TNF, in human proinflammatory M1 macrophages. TsSPs induce a concomitant anti-inflammatory M2 signature, with increased IL-10 production. Furthermore, they suppress CHIT activity and enhance secretion of matrix metalloproteinase 9. Short-term triggering of monocytes with TsSPs early during monocyte-to-macrophage differentiation imprinted these phenotypic alterations, suggesting long-lasting epigenetic changes. The TsSP-induced effects in M1 macrophages were completely reversed by inhibiting histone deacetylases, which corresponded with decreased histone acetylation at the TNF and IL6 promoters. These results demonstrate that TsSPs have a potent and sustained immunomodulatory effect on human macrophage differentiation and polarization through epigenetic remodeling and provide new insights into the mechanisms by which helminths modulate human immune responses.-Hoeksema, M. A., Laan, L. C., Postma, J. J., Cummings, R. D., de Winther, M. P. J., Dijkstra, C. D., van Die, I., Kooij, G. Treatment with Trichuris suis soluble products during monocyte-to-macrophage differentiation reduces inflammatory responses through epigenetic remodeling.

Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

GM-CSF promotes migration of human monocytes across the blood brain barrier.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Infiltration of monocytes into the CNS is crucial for disease onset and progression. Animal studies indicate that granulocyte-macrophages colony-stimulating factor (GM-CSF) may play an essential role in this process, possibly by acting on the migratory capacities of myeloid cells across the blood-brain barrier. This study describes the effect of GM-CSF on human monocytes, macrophages, and microglia. Furthermore, the expression of GM-CSF and its receptor was investigated in the CNS under healthy and pathological conditions. We show that GM-CSF enhances monocyte migration across human blood-brain barrier endothelial cells in vitro. Next, immunohistochemical analysis on human brain tissues revealed that GM-CSF is highly expressed by microglia and macrophages in MS lesions. The GM-CSF receptor is expressed by neurons in the rim of combined gray/white matter lesions and astrocytes. Finally, the effect of GM-CSF on human macrophages was determined, revealing an intermediate activation status, with a phenotype similar to that observed in active MS lesions. Together our data indicate that GM-CSF is a powerful stimulator of monocyte migration, and is abundantly present in the inflamed CNS where it may act as an activator of macrophages and microglia.

Activation status of human microglia is dependent on lesion formation stage and remyelination in multiple sclerosis.

Similar to macrophages, microglia adopt diverse activation states and contribute to repair and tissue damage in multiple sclerosis. Using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, we show that in vitro M1-polarized (proinflammatory) human adult microglia express the distinctive markers CD74, CD40, CD86, and CCR7, whereas M2 (anti-inflammatory) microglia express mannose receptor and the anti-inflammatory cytokine CCL22. The expression of these markers was assessed in clusters of activated microglia in normal-appearing white matter (preactive lesions) and areas of remyelination, representing reparative multiple sclerosis lesions. We show that activated microglia in preactive and remyelinating lesions express CD74, CD40, CD86, and the M2 markers CCL22 and CD209, but not mannose receptor. To examine whether this intermediate microglia profile is static or dynamic and thus susceptible to changes in the microenvironment, we polarized microglia into M1 or M2 phenotype in vitro and then subsequently treated them with the opposing polarization regimen. These studies revealed that expression of CD40, CXCL10, and mannose receptor is dynamic and that microglia, like macrophages, can switch between M1 and M2 phenotypic profiles. Taken together, our data define the differential activation states of microglia during lesion development in multiple sclerosis-affected CNS tissues and underscore the plasticity of human adult microglia in vitro.

Three distinct subsets of thymic epithelial cells in rats and mice defined by novel antibodies.

AIM: Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells. RESULTS: Rat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/-keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED21-K5-K8+Ulex europaeus lectin 1 (UEA-1)+CD205- medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-1-CD205- medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18+ED19+/-K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area. CONCLUSION: Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.

Human macrophage polarization in vitro: maturation and activation methods compared.

Macrophages form a heterogeneous cell population displaying multiple functions, and can be polarized into pro- (M1) or anti-inflammatory (M2) macrophages, by environmental factors. Their activation status reflects a beneficial or detrimental role in various diseases. Currently several in vitro maturation and activation protocols are used to induce an M1 or M2 phenotype. Here, the impact of different maturation factors (NHS, M-CSF, or GM-CSF) and activation methods (IFN-γ/LPS, IL-4, dexamethason, IL-10) on the macrophage phenotype was determined. Regarding macrophage morphology, pro-inflammatory (M1) activation stimulated cell elongation, and anti-inflammatory (M2) activation induced a circular appearance. Activation with pro-inflammatory mediators led to increased CD40 and CD64 expression, whereas activation with anti-inflammatory factors resulted in increased levels of MR and CD163. Production of pro-inflammatory cytokines was induced by activation with IFN-γ/LPS, and TGF-ß production was enhanced by the maturation factors M-CSF and GM-CSF. Our data demonstrate that macrophage marker expression and cytokine production in vitro is highly dependent on both maturation and activation methods. In vivo macrophage activation is far more complex, since a plethora of stimuli are present. Hence, defining the macrophage activation status ex vivo on a limited number of markers could be indecisive. From this study we conclude that maturation with M-CSF or GM-CSF induces a moderate anti- or pro-inflammatory state respectively, compared to maturation with NHS. CD40 and CD64 are the most distinctive makers for human M1 and CD163 and MR for M2 macrophage activation and therefore can be helpful in determining the activation status of human macrophages ex vivo.

Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation.

BACKGROUND: In neuroinflammatory diseases, macrophages can play a dual role in the process of tissue damage, depending on their activation status (M1 / M2). M1 macrophages are considered to exert damaging effects to neurons, whereas M2 macrophages are reported to aid regeneration and repair of neurons. Their migration within the central nervous system may be of critical importance in the final outcome of neurodegeneration in neuroinflammatory diseases e.g. multiple sclerosis (MS). To provide insight into this process, we examined the migratory capacity of human monocyte-derived M1 and M2 polarised macrophages towards chemoattractants, relevant for neuroinflammatory diseases like MS. METHODS: Primary cultures of human monocyte-derived macrophages were exposed to interferon gamma and lipopolysaccharide (LPS) to evoke proinflammatory (M1) activation or IL-4 to evoke anti-inflammatory (M2) activation. In a TAXIScan assay, migration of M0, M1 and M2 towards chemoattractants was measured and quantified. Furthermore the adhesion capacity and the expression levels of integrins as well as chemokine receptors of M0, M1 and M2 were assessed. Alterations in cell morphology were analysed using fluorescent labelling of the cytoskeleton. RESULTS: Significant differences were observed between M1 and M2 macrophages in the migration towards chemoattractants. We show that M2 macrophages migrated over longer distances towards CCL2, CCL5, CXCL10, CXCL12 and C1q compared to non-activated (M0) and M1 macrophages. No differences were observed in the adhesion of M0, M1 and M2 macrophages to multiple matrix components, nor in the expression of integrins and chemokine receptors. Significant changes were observed in the cytoskeleton organization upon stimulation with CCL2, M0, M1 and M2 macrophages adopt a spherical morphology and the cytoskeleton is rapidly rearranged. M0 and M2 macrophages are able to form filopodia, whereas M1 macrophages only adapt a spherical morphology. CONCLUSIONS: Together our results indicate that the alternative activation status of macrophages promotes their migratory properties to chemoattractants relevant for neuroinflammatory diseases like MS. Conversely, classically activated, proinflammatory macrophages have reduced migratory properties. Based on our results, we postulate that the activation status of the macrophage influences the capacity of the macrophages to rearrange their cytoskeleton. This is the first step in understanding how modulation of macrophage activation affects macrophage migration in neuroinflammatory diseases like MS.

Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status.

BACKGROUND: Macrophages play a dual role in multiple sclerosis (MS) pathology. They can exert neuroprotective and growth promoting effects but also contribute to tissue damage by production of inflammatory mediators. The effector function of macrophages is determined by the way they are activated. Stimulation of monocyte-derived macrophages in vitro with interferon-γ and lipopolysaccharide results in classically activated (CA/M1) macrophages, and activation with interleukin 4 induces alternatively activated (AA/M2) macrophages. METHODS: For this study, the expression of a panel of typical M1 and M2 markers on human monocyte derived M1 and M2 macrophages was analyzed using flow cytometry. This revealed that CD40 and mannose receptor (MR) were the most distinctive markers for human M1 and M2 macrophages, respectively. Using a panel of M1 and M2 markers we next examined the activation status of macrophages/microglia in MS lesions, normal appearing white matter and healthy control samples. RESULTS: Our data show that M1 markers, including CD40, CD86, CD64 and CD32 were abundantly expressed by microglia in normal appearing white matter and by activated microglia and macrophages throughout active demyelinating MS lesions. M2 markers, such as MR and CD163 were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. Double staining with anti-CD40 and anti-MR revealed that approximately 70% of the CD40-positive macrophages in MS lesions also expressed MR, indicating that the majority of infiltrating macrophages and activated microglial cells display an intermediate activation status. CONCLUSIONS: Our findings show that, although macrophages in active MS lesions predominantly display M1 characteristics, a major subset of macrophages have an intermediate activation status.

Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types.

BACKGROUND: Macrophages play an important role in neuroinflammatory diseases such as multiple sclerosis (MS) and spinal cord injury (SCI), being involved in both damage and repair. The divergent effects of macrophages might be explained by their different activation status: classically activated (CA/M1), pro-inflammatory, macrophages and alternatively activated (AA/M2), growth promoting, macrophages. Little is known about the effect of macrophages with these phenotypes in the central nervous system (CNS) and how they influence pathogenesis. The aim of this study was therefore to determine the characteristics of these phenotypically different macrophages in the context of the CNS in an in vitro setting. RESULTS: Here we show that bone marrow derived CA and AA macrophages have a distinct migratory capacity towards medium conditioned by various cell types of the CNS. AA macrophages were preferentially attracted by the low weight (< 10 kD) fraction of neuronal conditioned medium, while CA macrophages were attracted in higher numbers by astrocyte- and oligodendrocyte conditioned medium. Intrinsic motility was twice as high in AA macrophages compared to CA macrophages. The adhesion to extracellular matrix molecules (ECM) was significantly enhanced in CA macrophages compared to control and AA macrophages. The actin cytoskeleton was differentially organized between CA and AA macrophages, possibly due to greater activity of the GTPases RhoA and Rac in CA macrophages. Phagocytosis of myelin and neuronal fragments was increased in CA macrophages compared to AA macrophages. The increase in myelin phagocytosis was associated with higher expression of CR3/MAC-1 in CA macrophages. CONCLUSION: In conclusion, since AA macrophages are more motile and are attracted by NCM, they are prone to migrate towards neurons in the CNS. CA macrophages have a lower motility and a stronger adhesion to ECM. In neuroinflammatory diseases the restricted migration and motility of CA macrophages might limit lesion size due to bystander damage.

Adenosine triphosphate-binding cassette transporters mediate chemokine (C-C motif) ligand 2 secretion from reactive astrocytes: relevance to multiple sclerosis pathogenesis.

Adenosine triphosphate-binding cassette efflux transporters are highly expressed at the blood-brain barrier and actively hinder passage of harmful compounds, thereby maintaining brain homoeostasis. Since, adenosine triphosphate-binding cassette transporters drive cellular exclusion of potential neurotoxic compounds or inflammatory molecules, alterations in their expression and function at the blood-brain barrier may contribute to the pathogenesis of neuroinflammatory disorders, such as multiple sclerosis. Therefore, we investigated the expression pattern of different adenosine triphosphate-binding cassette efflux transporters, including P-glycoprotein, multidrug resistance-associated proteins-1 and -2 and breast cancer resistance protein in various well-characterized human multiple sclerosis lesions. Cerebrovascular expression of P-glycoprotein was decreased in both active and chronic inactive multiple sclerosis lesions. Interestingly, foamy macrophages in active multiple sclerosis lesions showed enhanced expression of multidrug resistance-associated protein-1 and breast cancer resistance protein, which coincided with their increased function of cultured foamy macrophages. Strikingly, reactive astrocytes display an increased expression of P-glycoprotein and multidrug resistance-associated protein-1 in both active and inactive multiple sclerosis lesions, which correlated with their enhanced in vitro activity on astrocytes derived from multiple sclerosis lesions. To investigate whether adenosine triphosphate-binding cassette transporters on reactive astrocytes can contribute to the inflammatory process, primary cultures of reactive human astrocytes were generated through activation of Toll-like receptor-3 to mimic the astrocytic phenotype as observed in multiple sclerosis lesions. Notably, blocking adenosine triphosphate-binding cassette transporter activity on reactive astrocytes inhibited immune cell migration across a blood-brain barrier model in vitro, which was due to the reduction of astrocytic release of the chemokine (C-C motif) ligand 2. Our data point towards a novel (patho)physiological role for adenosine triphosphate-binding cassette transporters, suggesting that limiting their activity by dampening astrocyte activation may open therapeutic avenues to diminish tissue damage during multiple sclerosis pathogenesis.

Magnetic resonance imaging of monocytes labeled with ultrasmall superparamagnetic particles of iron oxide using magnetoelectroporation in an animal model of multiple sclerosis.

AbstractInfiltrated monocytes play a crucial role in the demyelination process during multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS). Still, methods to monitor their infiltration pattern over time are lacking. In this study, magnetoelectroporation (MEP) was used to label rat monocytes with the superparamagnetic iron oxide particles Sinerem, Endorem, and Supravist. Supravist-labeled monocytes were injected in rats that we induced with experimental autoimmune encephalomyelitis, a model for MS. Imaging at 4.7 and 9.4 T revealed multiple foci of decreased signal intensity predominantly located in the cerebellum. Immunohistochemical evaluation confirmed the presence of intracellular iron in infiltrated cells, indicating the suitability of MEP to specifically follow labeled monocytes in vivo in this disease model. This technique may be further optimized and potentially used in MS patients to assess monocyte migration into the brain and to monitor the efficacy of therapeutic agents aimed at blocking cellular migration into the CNS.