The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells.

Clinical trials have shown that administration of the nematode Trichuris suis can be beneficial in treating various immune disorders. To provide insight into the mechanisms by which this worm suppresses inflammatory responses, an active component was purified from T. suis soluble products (TsSPs) that suppress---- TNF and IL-12 secretion from LPS-activated human dendritic cells (DCs). Analysis by liquid chromatography tandem mass spectrometry identified this compound as prostaglandin (PG)E2. The purified compound showed similar properties compared with TsSPs and commercial PGE2 in modulating LPS-induced expression of many cytokines and chemokines and in modulating Rab7B and P2RX7 expression in human DCs. Furthermore, the TsSP-induced reduction of TNF secretion from DCs is reversed by receptor antagonists for EP2 and EP4, indicating PGE2 action. T. suis secretes extremely high amounts of PGE2 (45-90 ng/mg protein) within their excretory/secretory products but few related lipid mediators as established by metabololipidomic analysis. Culture of T. suis with several cyclooxygenase (COX) inhibitors that inhibit mammalian prostaglandin synthesis affected the worm's motility but did not inhibit PGE2 secretion, suggesting that the worms can synthesize PGE2 via a COX-independent pathway. We conclude that T. suis secretes PGE2 to suppress proinflammatory responses in human DCs, thereby modulating the host's immune response.-Laan, L. C., Williams, A. R., Stavenhagen, K., Giera, M., Kooij, G., Vlasakov, I., Kalay, H., Kringel, H., Nejsum, P., Thamsborg, S. M., Wuhrer, M., Dijkstra, C. D., Cummings, R. D., van Die, I. The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells.

Serotonin: A mediator of the gut-brain axis in multiple sclerosis.

BACKGROUND: The significance of the gut microbiome for the pathogenesis of multiple sclerosis (MS) has been established, although the underlying signaling mechanisms of this interaction have not been sufficiently explored. OBJECTIVES: We address this point and use serotonin (5-hydroxytryptamine (5-HT))-a microbial-modulated neurotransmitter (NT) as a showcase to demonstrate that NTs regulated by the gut microbiome are potent candidates for mediators of the gut-brain axis in demyelinating disorders. Methods, Results, and Conclusion: Our comprehensive overview of literature provides evidence that 5-HT levels in the gut are controlled by the microbiome, both via secretion and through regulation of metabolites. In addition, we demonstrate that the gut microbiome can influence the formation of the serotonergic system (SS) in the brain. We also show that SS alterations have been related to MS directly-altered expression of 5-HT transporters in central nervous system (CNS) and indirectly-beneficial effects of 5-HT modulating drugs on the course of the disease and higher prevalence of depression in patients with MS. Finally, we discuss briefly the role of other microbiome-modulated NTs such as γ-aminobutyric acid and dopamine in MS to highlight a new direction for future research aiming to relate microbiome-regulated NTs to demyelinating disorders.

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.

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.

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.

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.

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.

Mutant TP53 switches therapeutic vulnerability during gastric cancer progression within interleukin-6 family cytokines.

Although aberrant activation of the KRAS and PI3K pathway alongside TP53 mutations account for frequent aberrations in human gastric cancers, neither the sequence nor the individual contributions of these mutations have been clarified. Here, we establish an allelic series of mice to afford conditional expression in the glandular epithelium of KrasG12D;Pik3caH1047R or Trp53R172H and/or ablation of Pten or Trp53. We find that KrasG12D;Pik3caH1047R is sufficient to induce adenomas and that lesions progress to carcinoma when also harboring Pten deletions. An additional challenge with either Trp53 loss- or gain-of-function alleles further accelerated tumor progression and triggered metastatic disease. While tumor-intrinsic STAT3 signaling in response to gp130 family cytokines remained as a gatekeeper for all stages of tumor development, metastatic progression required a mutant Trp53-induced interleukin (IL)-11 to IL-6 dependency switch. Consistent with the poorer survival of patients with high IL-6 expression, we identify IL-6/STAT3 signaling as a therapeutic vulnerability for TP53-mutant gastric cancer.

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.

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.

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.

Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis.

Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood-borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well-characterized antibodies to axonal antigens, that axonal damage is associated with HLA-DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA-DR(+) cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF-L), present in white and gray matter, was observed in human microglia. The number of NF-L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF-L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF-L is taken up and degraded by the cell. In vivo, NF-L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA-DR(+) cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens.

Fingolimod attenuates ceramide-induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes.

Alterations in sphingolipid metabolism are described to contribute to various neurological disorders. We here determined the expression of enzymes involved in the sphingomyelin cycle and their products in postmortem brain tissue of multiple sclerosis (MS) patients. In parallel, we investigated the effect of the sphingosine-1 receptor agonist Fingolimod (Gilenya(®)) on sphingomyelin metabolism in reactive astrocytes and determined its functional consequences for the process of neuro-inflammation. Our results demonstrate that in active MS lesions, marked by large number of infiltrated immune cells, an altered expression of enzymes involved in the sphingomyelin cycle favors enhanced ceramide production. We identified reactive astrocytes as the primary cellular source of enhanced ceramide production in MS brain samples. Astrocytes isolated from MS lesions expressed enhanced mRNA levels of the ceramide-producing enzyme acid sphingomyelinase (ASM) compared to astrocytes isolated from control white matter. In addition, TNF-α treatment induced ASM mRNA and ceramide levels in astrocytes isolated from control white matter. Incubation of astrocytes with Fingolimod prior to TNF-α treatment reduced ceramide production and mRNA expression of ASM to control levels in astrocytes. Importantly, supernatants derived from reactive astrocytes treated with Fingolimod significantly reduced transendothelial monocyte migration. Overall, the present study demonstrates that reactive astrocytes represent a possible additional cellular target for Fingolimod in MS by directly reducing the production of pro-inflammatory lipids and limiting subsequent transendothelial leukocyte migration.

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.

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.

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.

The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria.

The plasma membrane glycoprotein receptor CD163 is a member of the scavenger receptor cystein-rich (SRCR) superfamily class B that is highly expressed on resident tissue macrophages in vivo. Previously, the molecule has been shown to act as a receptor for hemoglobin-haptoglobin complexes and to mediate cell-cell interactions between macrophages and developing erythroblasts in erythroblastic islands. Here, we provide evidence for a potential role for CD163 in host defense. In particular, we demonstrate that CD163 can function as a macrophage receptor for bacteria. CD163 was shown to bind both Gram-positive and -negative bacteria, and a previously identified cell-binding motif in the second scavenger domain of CD163 was sufficient to mediate this binding. Expression of CD163 in monocytic cells promoted bacteria-induced proinflammatory cytokine production. Finally, newly generated antagonistic antibodies against CD163 were able to potently inhibit cytokine production elicited by bacteria in freshly isolated human monocytes. These findings identify CD163 as a macrophage receptor for bacteria and suggest that, during bacterial infection, CD163 on resident tissue macrophages acts as an innate immune sensor and inducer of local inflammation.

Sphingosine 1-phosphate receptor 1 and 3 are upregulated in multiple sclerosis lesions.

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1-phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well-characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S1P(1/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.

Flavonoids influence monocytic GTPase activity and are protective in experimental allergic encephalitis.

In the chronic disabling disease multiple sclerosis (MS), migration of monocytes across the blood-brain barrier is a crucial step in the formation of new lesions in the central nervous system (CNS). Infiltrating monocyte-derived macrophages secrete inflammatory mediators such as oxygen radicals, which contribute to axonal demyelination and damage, resulting in neurological deficits. Flavonoids are compounds occurring naturally in food, which scavenge oxygen radicals and have antiinflammatory properties. To investigate whether they might suppress clinical symptoms in MS, we treated rats sensitized for acute and chronic experimental allergic encephalomyelitis, an experimental model of MS, with flavonoids. We demonstrated that the flavonoid luteolin substantially suppressed clinical symptoms and prevented relapse when administered either before or after disease onset. Luteolin treatment resulted in reduced inflammation and axonal damage in the CNS by preventing monocyte migration across the brain endothelium. Luteolin influenced migration by modulating the activity of Rho GTPases, signal transducers involved in transendothelial migration. Oral administration of luteolin also significantly reduced clinical symptoms.