Pre-treatment with the viral Toll-like receptor 3 agonist poly(I:C) modulates innate immunity and protects neutropenic mice infected intracerebrally with Escherichia coli.

BACKGROUND: Individuals with impaired immunity are more susceptible to infections than immunocompetent subjects. No vaccines are currently available to induce protection against E. coli meningoencephalitis. This study evaluated the potential of poly(I:C) pre-treatment to induce trained immunity. Poly(I:C) was administered as a non-specific stimulus of innate immune responses to protect immunocompetent and neutropenic wild-type mice from a subsequent challenge by the intracranial injection of E. coli K1. METHODS: Three days prior to infection, mice received an intraperitoneal injection of poly(I:C) or vehicle. Kaplan-Meier survival curves were analyzed. In short-term experiments, bacterial titers and the inflammatory response were characterized in the blood, cerebellum, and spleen homogenates. NK cell subpopulations in the brain and spleen were analyzed by flow cytometry. Numbers of microglia and activation scores were evaluated by histopathology. RESULTS: Pre-treatment with 200 µg poly(I:C) increased survival time, reduced mortality, and enhanced bacterial clearance in the blood, cerebellum, and spleen at early infection in neutropenic mice. Poly(I:C)-mediated protection correlated with an augmented number of NK cells (CD45+NK1.1+CD3-) and Iba-1+ microglial cells and a higher production of IFN-γ in the brain. In the spleen, levels of CCL5/RANTES and IFN-γ were increased and sustained in surviving poly(I:C)-treated animals for 14 days after infection. In immunocompetent animals, survival time was not significantly prolonged in poly(I:C)-treated animals although poly(I:C) priming reduced brain bacterial concentrations compared with vehicle-injected animals at early infection. CONCLUSIONS: Pre-treatment with the viral TLR3 agonist poly(I:C) modulated innate immune responses and strengthened the resistance of neutropenic mice against E. coli K1 meningoencephalitis.

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ.

Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia-neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K(+) concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1ß, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30-100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.

Physiology of microglia.

Microglial cells are the resident macrophages in the central nervous system. These cells of mesodermal/mesenchymal origin migrate into all regions of the central nervous system, disseminate through the brain parenchyma, and acquire a specific ramified morphological phenotype termed "resting microglia." Recent studies indicate that even in the normal brain, microglia have highly motile processes by which they scan their territorial domains. By a large number of signaling pathways they can communicate with macroglial cells and neurons and with cells of the immune system. Likewise, microglial cells express receptors classically described for brain-specific communication such as neurotransmitter receptors and those first discovered as immune cell-specific such as for cytokines. Microglial cells are considered the most susceptible sensors of brain pathology. Upon any detection of signs for brain lesions or nervous system dysfunction, microglial cells undergo a complex, multistage activation process that converts them into the "activated microglial cell." This cell form has the capacity to release a large number of substances that can act detrimental or beneficial for the surrounding cells. Activated microglial cells can migrate to the site of injury, proliferate, and phagocytose cells and cellular compartments.

Activin A increases phagocytosis of Escherichia coli K1 by primary murine microglial cells activated by toll-like receptor agonists.

BACKGROUND: Bacterial meningitis is associated with high mortality and long-term neurological sequelae. Increasing the phagocytic activity of microglia could improve the resistance of the CNS against infections. We studied the influence of activin A, a member of the TGF-ß family with known immunoregulatory and neuroprotective effects, on the functions of microglial cells in vitro. METHODS: Primary murine microglial cells were treated with activin A (0.13 ng/ml-13 µg/ml) alone or in combination with agonists of TLR2, 4, and 9. Phagocytosis of Escherichia coli K1 as well as release of TNF-α, IL-6, CXCL1, and NO was assessed. RESULTS: Activin A dose-dependently enhanced the phagocytosis of Escherichia coli K1 by microglial cells activated by agonists of TLR2, 4, and 9 without further increasing NO and proinflammatory cytokine release. Cell viability of microglial cells was not affected by activin A. CONCLUSIONS: Priming of microglial cells with activin A could increase the elimination of bacteria in bacterial CNS infections. This preventive strategy could improve the resistance of the brain to infections, particularly in elderly and immunocompromised patients.

A presumed antagonistic LPS identifies distinct functional organization of TLR4 in mouse microglia.

Microglia as principle innate immune cells of the central nervous system (CNS) are the first line of defense against invading pathogens. They are capable of sensing infections through diverse receptors, such as Toll-like receptor 4 (TLR4). This receptor is best known for its ability to recognize bacterial lipopolysaccharide (LPS), a causative agent of gram-negative sepsis and septic shock. A putative, naturally occurring antagonist of TLR4 derives from the photosynthetic bacterium Rhodobacter sphaeroides. However, the antagonistic potential of R. sphaeroides LPS (Rs-LPS) is no universal feature, since several studies suggested agonistic rather than antagonistic actions of this molecule depending on the investigated mammalian species. Here we show the agonistic versus antagonistic potential of Rs-LPS in primary mouse microglia. We demonstrate that Rs-LPS efficiently induces the release of cytokines and chemokines, which depends on TLR4, MyD88, and TRIF, but not CD14. Furthermore, Rs-LPS is able to regulate the phagocytic capacity of microglia as agonist, while it antagonizes Re-LPS-induced MHC I expression. Finally, to our knowledge, we are the first to provide in vivo evidence for an agonistic potential of Rs-LPS, as it efficiently triggers the recruitment of peripheral immune cells to the endotoxin-challenged CNS. Together, our results argue for a versatile and complex organization of the microglial TLR4 system, which specifically translates exogenous signals into cellular functions. Importantly, as demonstrated here for microglia, the antagonistic potential of Rs-LPS needs to be considered with caution, as reactions to Rs-LPS not only differ by cell type, but even by function within one cell type.

Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system.

The action of type I interferons in the central nervous system (CNS) during autoimmunity is largely unknown. Here, we demonstrate elevated interferon beta concentrations in the CNS, but not blood, of mice with experimental autoimmune encephalomyelitis (EAE), a model for CNS autoimmunity. Furthermore, mice devoid of the broadly expressed type I IFN receptor (IFNAR) developed exacerbated clinical disease accompanied by a markedly higher inflammation, demyelination, and lethality without shifting the T helper 17 (Th17) or Th1 cell immune response. Whereas adoptive transfer of encephalitogenic T cells led to enhanced disease in Ifnar1(-/-) mice, newly created conditional mice with B or T lymphocyte-specific IFNAR ablation showed normal EAE. The engagement of IFNAR on neuroectodermal CNS cells had no protective effect. In contrast, absence of IFNAR on myeloid cells led to severe disease with an enhanced effector phase and increased lethality, indicating a distinct protective function of type I IFNs during autoimmune inflammation of the CNS.

Thioredoxins and Methionine Sulfoxide Reductases in the Pathophysiology of Pneumococcal Meningitis.

BACKGROUND: Pneumococcal proteins involved in the resistance against oxidative stress are present in all strains and therefore are potential antigens that could be suitable for new therapies and/or vaccines. Their role in the pathogenesis of pneumococcal meningitis has not been addressed. METHODS: We investigated the individual contributions of extracellular thioredoxin lipoproteins (Etrx1 and Etrx2) and the intracellular and extracellular methionine sulfoxide reductases (SpMsrAB1 and SpMsrAB2, respectively) in the progression and outcome of pneumococcal meningitis, using Kaplan-Meier survival curves, bacteriological and histological studies, and measurements of proinflammatory mediators. RESULTS: The absence of Etrx1, Etrx2, or SpMsrAB1 moderately attenuated virulence as compared to the wild-type strain but did not significantly affect bacterial growth in the brain and bloodstream. Loss of function of SpMsrAB2 alone, both Etrx proteins, or both SpMsrAB proteins resulted in a less severe course of infection, with low numbers of animals dying of infection, a lower risk of associated meningeal inflammation, and reduced bacterial densities in the cerebellum, blood, and spleen. CONCLUSIONS: Our data support the importance of the extracellular redox repair system in virulence and its potential as a target for the design of new antimicrobials and vaccine formulations against Streptococcus pneumoniae.

CD14 is a key organizer of microglial responses to CNS infection and injury.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury-associated factors. We show that its co-receptor CD14 serves three non-redundant functions in microglia. First, it confers an up to 100-fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon ß-mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage-associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo- or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non-TLR systems to thereby fine-tune microglial damage-sensing capacity upon infectious and non-infectious CNS challenges.

CD11c-positive cells from brain, spleen, lung, and liver exhibit site-specific immune phenotypes and plastically adapt to new environments.

The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double-positive cells from lung, liver, and spleen in healthy mice using seven-color flow cytometry. We identified unique, site-specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC-II. Furthermore, we observed the two known CD45-positive populations (CD45(high) and CD45(int) ) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45(high) population. CD11c-positive microglia lacked MHC-II expression and CD45(high) /CD11c-positive cells from the brain have a lower percentage of MHC-II-positive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC-II-positive splenocytes is paralleled by down-regulation of MHC-II, whereas brain-derived mononuclear cells neither ramified nor up-regulated MHC-II in OSSCs. Thus, brain-derived mononuclear cells maintain their MHC-II-negative phenotype within the environment of an immune organ. Intraparenchymal CD11c-positive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC-II expression.

Tyrphostin AG126 exerts neuroprotection in CNS inflammation by a dual mechanism.

The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.

Vitamin D deficiency decreases survival of bacterial meningoencephalitis in mice.

BACKGROUND: Meningoencephalitis caused by Escherichia coli is associated with high rates of mortality and risk of neurological sequelae in newborns and infants and in older or immunocompromised adults. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. METHODS: In vivo, we studied the effects of vitamin D3 on survival and the host's immune response in experimental bacterial meningoencephalitis in mice after intracerebral E. coli infection. To produce different systemic vitamin D3 concentrations, mice received a low, standard, or high dietary vitamin D3 supplementation. Bacterial titers in blood, spleen, and brain homogenates were determined. Leukocyte infiltration was assessed by histological scores, and tissue cytokine or chemokine concentrations were measured. RESULTS: Mice fed a diet with low vitamin D3 concentration died earlier than control animals after intracerebral infection. Vitamin D deficiency did not inhibit leukocyte recruitment into the subarachnoid space and did not lead to an increased density of bacteria in blood, spleen, or brain homogenates. The release of proinflammatory interleukin (IL)-6 was decreased and the release of anti-inflammatory IL-10 was increased in mice fed a diet with high vitamin D3 supplementation. CONCLUSION: Our observations suggest a detrimental role of vitamin D deficiency in bacterial central nervous system infections. Vitamin D may exert immune regulatory functions.

Vitamin d deficiency reduces the immune response, phagocytosis rate, and intracellular killing rate of microglial cells.

Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality and neurological sequelae. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. Vitamin D has potent effects on human immunity, including induction of antimicrobial peptides (AMPs) and suppression of T-cell proliferation, but its influence on microglial cells is unknown. The purpose of the present study was to determine the effects of vitamin D deficiency on the phagocytosis rate, intracellular killing, and immune response of murine microglial cultures after stimulation with the Toll-like receptor (TLR) agonists tripalmitoyl-S-glyceryl-cysteine (TLR1/2), poly(I·C) (TLR3), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9). Upon stimulation with high concentrations of TLR agonists, the release of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) was decreased in vitamin D-deficient compared to that in vitamin D-sufficient microglial cultures. Phagocytosis of E. coli K1 after stimulation of microglial cells with high concentrations of TLR3, -4, and -9 agonists and intracellular killing of E. coli K1 after stimulation with high concentrations of all TLR agonists were lower in vitamin D-deficient microglial cells than in the respective control cells. Our observations suggest that vitamin D deficiency may impair the resistance of the brain against bacterial infections.

Rho kinase inhibition modulates microglia activation and improves survival in a model of amyotrophic lateral sclerosis.

Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons (MN) and their axons, but is also influenced by neighboring cells such as astrocytes and microglial cells. The role of microglia in ALS is complex as it switches from an anti-inflammatory and neuroprotective phenotype in early disease to a proinflammatory and neurotoxic phenotype in later stages. Our previous studies in models of neurodegeneration identified rho kinase (ROCK) as a target, which can be manipulated to beneficially influence disease progression. Here, we examined the neuroprotective potential of the ROCK inhibitor Fasudil to target the central pathogenic features of ALS. Application of Fasudil to kainic acid-lesioned primary MN in vitro resulted in a strong prosurvival effect. In vivo, SOD1(G93A) mice benefited from oral treatment with Fasudil showing prolonged survival and improved motor function. These findings were correlated to an improved survival of motor neurons and a pronounced alteration of astroglial and microglial cell infiltration of the spinal cord under Fasudil treatment. Modeling a proinflammatory microglial phenotype by stimulation with LPS in vitro, Fasudil decreased the release of proinflammatory cytokines and chemokines TNFα, Il6, CCL2, CCL3, and CCL5 while CXCL1 release was only transiently suppressed. In sciatic nerve motor axons, neuromuscular junction remodeling processes were increased. In conclusion, we provide preclinical and neurobiological evidence that inhibition of ROCK by the clinically approved small molecule inhibitor Fasudil may be a novel therapeutic approach in ALS combining both neuroprotection and immunomodulation for the cure of this devastating disease.

Influence of intrathecal delivery of bone marrow-derived mesenchymal stem cells on spinal inflammation and pain hypersensitivity in a rat model of peripheral nerve injury.

BACKGROUND: Multipotent mesenchymal stem (stromal) cells (MSCs) have been credited with immunomodulative properties, supporting beneficial outcomes when transplanted into a variety of disease models involving inflammation. Potential mechanisms include the secretion of paracrine factors and the establishment of a neurotrophic microenvironment. To test the hypothesis that MSCs release soluble mediators that can attenuate local inflammation, we here analysed the influence of MSCs on the activation of microglia cells, as well as on inflammatory parameters and pain behaviour in a surgical rat model of neuropathic pain. METHODS: We focussed on an experimental model of partial sciatic nerve ligation (PSNL), characterised by a rapid and persistent inflammation in the dorsal lumbar spinal cord where sensory inputs from the sciatic nerve are processed. Via indwelling intrathecal catheters, MSCs were repetitively grafted into the intrathecal lumbar space. Animals were evaluated for mechanical and thermal hypersensitivity over a period of 21 days after PSNL. Afterwards, spinal cords were processed for immunohistochemical analysis of the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) and quantification of inflammatory markers in ipsilateral dorsal horns. We hypothesised that injections on postsurgical days 2 to 4 would interfere with microglial activation, leading to a reduced production of pro-inflammatory cytokines and amelioration of pain behaviour. RESULTS: PSNL-induced mechanical allodynia or heat hyperalgesia were not influenced by MSC transplantation, and spinal cord inflammatory processes remained largely unaffected. Indeed, the early microglial response to PSNL characterised by increased Iba1 expression in the lumbar dorsal horn was not significantly altered and cytokine levels in the spinal cord at 21 days after surgery were similar to those found in vehicle-injected animals. Grafted MSCs were detected close to the pia mater, but were absent within the spinal cord parenchyma. CONCLUSIONS: We conclude that intrathecal administration is not an appropriate route to deliver cells for treatment of acute spinal cord inflammation as it leads to entrapment of grafted cells within the pia mater. We propose that the early inflammatory response triggered by PSNL in the lumbar spinal cord failed to effectively recruit MSCs or was insufficient to disturb the tissue integrity so as to allow MSCs to penetrate the spinal cord parenchyma.

Intraperitoneal prophylaxis with CpG oligodeoxynucleotides protects neutropenic mice against intracerebral Escherichia coli K1 infection.

BACKGROUND: Prophylaxis with unmethylated cytosine phosphate guanidine (CpG) oligodeoxynucleotides (ODN) protects against several systemic experimental infections. Escherichia coli is a major cause of Gram-negative neonatal bacterial meningitis and also causes meningitis and meningoencephalitis in older and immunocompromised patients. METHODS: Wild-type (wt) and Toll-like receptor 9 (TLR9)-deficient mice were rendered neutropenic by intraperitoneal administration of the anti-Ly-6G monoclonal antibody. Immunocompetent and neutropenic mice received intraperitoneal CpG ODN or vehicle 72 h prior to induction of E. coli K1 meningoencephalitis. RESULTS: Pre-treatment with CpG ODN significantly increased survival of neutropenic wt mice from 33% to 75% (P = 0.0003) but did not protect neutropenic TLR9-/- mice. The protective effect of CpG ODN was associated with an enhanced production of interleukin (IL)-12/IL-23p40 with sustained increased levels in serum and spleen at least for 17 days after conditioning compared to buffer-treated animals. CpG-treated neutropenic wt mice showed reduced bacterial concentrations and increased recruitment of Ly6ChighCCR2+ monocytes in brain and spleen 42 h after infection. The levels of macrophage inflammatory protein 1α (MIP-1α) and interferon gamma (IFN-γ) in spleen were higher 42 h after infection in CpG-treated compared to buffer-treated neutropenic animals. In immunocompetent mice, prophylaxis with CpG ODN did not significantly increase survival compared to the buffer group (60% vs. 45%, P = 0.2). CONCLUSIONS: These findings suggest that systemic administration of CpG ODN may help to prevent bacterial CNS infections in immunocompromised individuals.

Resistance of the brain to Escherichia coli K1 infection depends on MyD88 signaling and the contribution of neutrophils and monocytes.

Escherichia coli is the leading cause of Gram-negative neonatal bacterial meningitis and also causes meningitis and meningoencephalitis in older and immunocompromised patients. Here, we determined the contribution of granulocytes, monocytes, and TLR signaling cascades in the resistance of adult mice to Escherichia coli K1 brain infection. Deficiency in MyD88 (myd88(-/-)) but not in TRIF (trif(lps2)) adaptor proteins dramatically reduced the survival of animals. Depletion of CD11b(+) Ly-6G(+) Ly-6C(int) neutrophils by application of the anti-Ly-6G (1A8) monoclonal antibody (MAb) led to higher bacterial loads in cerebellum and spleen tissue and resulted in increased mortality compared to those of isotype-treated controls. Depletion of CD11b(+) Ly-6G(+) Ly-6C(int) neutrophils and CD11b(+) Ly-6G(-) Ly-6C(high) monocytes by administration of the anti-Gr-1 (RB6-8C5) MAb rendered mice even more susceptible to the infection, with higher central nervous system (CNS) and spleen bacterial burdens than anti-Ly-6G-treated animals. Depletion of ∼50% of CD11b(+) Ly-6G(-) Ly-6C(high) monocytes by injection of the anti-CCR2 (MC-21) MAb resulted in a trend toward higher mortality compared to that with isotype treatment. Production of interleukin 1ß (IL-1ß), IL-6, KC, and MIP-2 in the CNS strongly depended on the bacterial load: increased levels of these cytokines/chemokines were found after depletion of CD11b(+) Ly-6G(+) Ly-6C(int) neutrophils alone or together with CD11b(+) Ly-6G(-) Ly-6C(high) monocytes. These findings identify Toll-like receptor (TLR)-MyD88 signaling and neutrophil and monocyte activity as critical elements in the early host defense against E. coli meningitis.

Carcinoma cells misuse the host tissue damage response to invade the brain.

The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis.

Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis.

The transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenesis of autoimmune diseases. Here, we show that oligodendrocytes secrete small membrane vesicles called exosomes, which are specifically and efficiently taken up by microglia both in vitro and in vivo. Internalisation of exosomes occurs by a macropinocytotic mechanism without inducing a concomitant inflammatory response. After stimulation of microglia with interferon-γ, we observe an upregulation of MHC class II in a subpopulation of microglia. However, exosomes are preferentially internalised in microglia that do not seem to have antigen-presenting capacity. We propose that the constitutive macropinocytotic clearance of exosomes by a subset of microglia represents an important mechanism through which microglia participate in the degradation of oligodendroglial membrane in an immunologically 'silent' manner. By designating the capacity for macropinocytosis and antigen presentation to distinct cells, degradation and immune function might be assigned to different subtypes of microglia.

Empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the TLR1/2 agonist Pam3CSK 4 (BLP).

Liposomes are frequently used in cancer therapy to encapsulate and apply anticancer drugs. Here, we show that a systemic treatment of mice bearing skin tumors with empty phosphatidylcholine liposomes (PCL) resulted in inhibition of tumor growth, which was similar to that observed with the synthetic bacterial lipoprotein and TLR1/2 agonist Pam(3)CSK(4) (BLP). Both compounds led to a substantial decrease of macrophages in spleen and in the tumor-bearing skin. Furthermore, both treatments induced the expression of typical macrophage markers in the tumor-bearing tissue. As expected, BLP induced the expression of the M1 marker genes Cxcl10 and iNOS, whereas PCL, besides inducing iNOS, also increased the M2 marker genes Arg1 and Trem2. In vitro experiments demonstrated that neither PCL nor BLP influenced proliferation or survival of tumor cells, whereas both compounds inhibited proliferation and survival and increased the migratory capacity of bone marrow-derived macrophages (BMDM). However, in contrast to BLP, PCL did not activate cytokine secretion and induced a different BMDM phenotype. Together, the data suggest that similar to BLP, PCL induce an antitumor response by influencing the tumor microenvironment, in particular by functional alterations of macrophages, however, in a distinct manner from those induced by BLP.

Histone deacetylase inhibitors suppress immune activation in primary mouse microglia.

Neuroinflammation is required for tissue clearance and repair after infections or insults. To prevent excessive damage, it is crucial to limit the extent of neuroinflammation and thereby the activation of its principal effector cell, microglia. The two main major innate immune cell types in the CNS are astrocytes and microglia. Histone deacetylases (HDACs) have been implicated in regulating the innate inflammatory response, and here we addressed their role in pure astrocyte and microglia cultures. Endogenous HDAC expression levels were determined in microglia and astrocytes and after treatment with lipopolysaccharide (LPS) or LPS and interferon γ (IFNγ). The relative expression level of HDACs was reduced in LPS- or LPS/IFNγ (with the exception of HDAC1 and -7)-stimulated astrocytes and increased in microglia after LPS treatment both in primary cultures and in microglia acutely isolated from LPS-treated mice, so we focused on the inflammatory response in microglia. Primary microglia cultures were treated with LPS in the presence or absence of HDAC inhibitors (HDACi). Expression and release of inflammatory cytokines was determined by quantitative RT-PCR, flow cytometry, and ELISA. HDACi strongly suppressed LPS-induced cytokine expression and release by microglia. Furthermore, expression of M1- and M2-associated activation markers was suppressed, and the migratory behavior of microglia was attenuated. Our findings strongly suggest that HDACi suppress innate immune activation in microglia.