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.

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.

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.

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.

Toll-like receptor activation reveals developmental reorganization and unmasks responder subsets of microglia.

The sentinel and immune functions of microglia require rapid and appropriate reactions to infection and damage. Their Toll-like receptors (TLRs) sense both as threats. However, whether activated microglia mount uniform responses or whether subsets conduct selective tasks is unknown. We demonstrate that murine microglia reorganize their responses to TLR activations postnatally and that this process comes with a maturation of TLR4-organized functions. Although induction of MHCI for antigen presentation remains as a pan-populational feature, synthesis of TNFα becomes restricted to a subset, even within adult central nervous system regions. Response heterogeneity is evident ex vivo, in situ, and in vivo, but is not limited to TNFα production or to TLR-triggered functions. Also, clearance activities for myelin under physiological and pathophysiological conditions, IFNγ-enforced upregulation of MHCII, or challenged inductions of other proinflammatory factors reveal dissimilar microglial contributions. Notably, response heterogeneity is also confirmed in human brain tissue. Our findings suggest that microglia divide by constitutive and inducible capacities. Privileged production of inflammatory mediators assigns a master control to subsets. Sequestration of clearance of endogenous material versus antigen presentation in exclusive compartments can separate potentially interfering functions. Finally, subsets rather than a uniform population of microglia may assemble the reactive phenotypes in responses during infection, injury, and rebuilding, warranting consideration in experimental manipulation and therapeutic strategies.

Reduced astrocytic NF-κB activation by laquinimod protects from cuprizone-induced demyelination.

Laquinimod (LAQ) is a new oral immunomodulatory compound that reduces relapse rate, brain atrophy and disability progression in multiple sclerosis (MS). LAQ has well-documented effects on inflammation in the periphery, but little is known about its direct activity within the central nervous system (CNS). To elucidate the impact of LAQ on CNS-intrinsic inflammation, we investigated the effects of LAQ on cuprizone-induced demyelination in mice in vivo and on primary CNS cells in vitro. Demyelination, inflammation, axonal damage and glial pathology were evaluated in LAQ-treated wild type and Rag-1-deficient mice after cuprizone challenge. Using primary cells we tested for effects of LAQ on oligodendroglial survival as well as on cytokine secretion and NF-κB activation in astrocytes and microglia. LAQ prevented cuprizone-induced demyelination, microglial activation, axonal transections, reactive gliosis and oligodendroglial apoptoses in wild type and Rag-1-deficient mice. LAQ significantly decreased pro-inflammatory factors in stimulated astrocytes, but not in microglia. Oligodendroglial survival was not affected by LAQ in vitro. Astrocytic, but not microglial, NF-κB activation was markedly reduced by LAQ as evidenced by NF-κB reporter assay. LAQ also significantly decreased astrocytic NF-κB activation in cuprizone-treated mice. Our data indicate that LAQ prevents cuprizone-induced demyelination by attenuating astrocytic NF-κB activation. These effects are CNS-intrinsic and not mediated by peripheral immune cells. Therefore, LAQ downregulation of the astrocytic pro-inflammatory response may be an important mechanism underlying its protective effects on myelin, oligodendrocytes and axons. Modulation of astrocyte activation may be an attractive therapeutic target to prevent tissue damage in MS.

Intralesional TLR4 agonist treatment strengthens the organ defense against colonizing cancer cells in the brain.

Brain metastasis in breast cancer remains difficult to treat and its incidence is increasing. Therefore, the development of new therapies is of utmost clinical relevance. Recently, toll-like receptor (TLR) 4 was correlated with IL6 expression and poor prognosis in 1 215 breast cancer primaries. In contrast, we demonstrated that TLR4 stimulation reduces microglia-assisted breast cancer cell invasion. However, the expression, prognostic value, or therapeutic potential of TLR signaling in breast cancer brain metastasis have not been investigated. We thus tested the prognostic value of various TLRs in two brain-metastasis gene sets. Furthermore, we investigated different TLR agonists, as well as MyD88 and TRIF-deficient microenvironments in organotypic brain-slice ex vivo co-cultures and in vivo colonization experiments. These experiments underline the ambiguous roles of TLR4, its adapter MyD88, and the target nitric oxide (NO) during brain colonization. Moreover, analysis of the gene expression datasets of breast cancer brain metastasis patients revealed associations of TLR1 and IL6 with poor overall survival. Finally, our finding that a single LPS application at the onset of colonization shapes the later microglia/macrophage reaction at the macro-metastasis brain-parenchyma interface (MMPI) and reduces metastatic infiltration into the brain parenchyma may prove useful in immunotherapeutic considerations.

CD14 and TRIF govern distinct responsiveness and responses in mouse microglial TLR4 challenges by structural variants of LPS.

Toll-like receptor (TLR) 4 responds to a range of agonists in infection and injury, but is best known for the recognition of bacterial lipopolysaccharides (LPS). Assembly in heterologous receptor complexes as well as signaling through both MyD88 and TRIF adaptor proteins, as unmatched by other TLRs, could underlie its versatile response options, probably also in a cell type-dependent manner. We show that microglia, the CNS macrophages, react to diverse LPS variants, including smooth (S) and rough (R) LPS chemotypes, with cytokine/chemokine induction, MHC I expression and suppression of myelin phagocytosis. The TLR4 co-receptor CD14 was shown in peritoneal macrophages to be essential for S-LPS effects and the link of both S- and R-LPS to TRIF signaling. In contrast, cd14(-/-) microglia readily respond to S- and R-LPS, suggesting an a priori high(er) sensitivity to both chemotypes, while CD14 confers increased S- and R-LPS potencies and compensates for their differences. Importantly, CD14 controls the magnitude and shapes the profile of cyto/chemokine production, this influence being itself regulated by critical LPS concentrations. Comparing reactive phenotypes of microglia with deficiencies in CD14, MyD88 and TRIF (cd14(-/-), myd88(-/-), and trif(lps2)), we found that distinct signaling routes organize for individual functions in either concerted or non-redundant fashion and that CD14 has contributions beyond the link to TRIF. Modulation of response profiles by key cytokines finally reveals that the microglial TLR4 can differentiate between the class of LPS structures and a self-derived agonist, fibronectin. It thus proves as a sophisticated decision maker in infectious and non-infectious CNS challenges.

Microglia promote colonization of brain tissue by breast cancer cells in a Wnt-dependent way.

Although there is increasing evidence that blood-derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf-2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2-like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor-educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy.

Expression of coagulation factors and their receptors in tumor tissue and coagulation factor upregulation in peripheral blood of patients with cerebral carcinoma metastases.

BACKGROUND: Patients with malignancies often suffer from thrombembolic events that complicate the course of cancer disease and reduce the patients' quality of life or shorten the survival time in severe cases. This phenomenon is also known for patients with primary or secondary brain tumors; but the reasons are not identified. METHODS: We performed a prospective case-controlled study of patients with brain metastases but without any active peripheral tumor site. Blood of patients was collected perioperatively and investigated for coagulation factor activities. Moreover, we analyzed the expression of coagulation factors and their receptors within the tumor material of brain metastases from clear-cell renal cell carcinomas and small-cell carcinomas of the lung. RESULTS: Here, we show that even patients without an active peripheral tumor disease that means without any tumor masses outside the central nervous system after anticancer treatment by surgery, radiation therapy, or chemotherapy but with symptomatic brain metastasis develop an increased systemic activation of multiple coagulation factors. The pro-coagulatory state is expressed preoperatively, but also can be observed in the early postoperative period. Additionally to that, intracerebral metastases of clear-cell renal cell carcinomas and of small-cell carcinomas of the lung express prothrombin, thrombin, factor X, and the protease-activated receptors type 1, 2, 3, and 4. CONCLUSIONS: These observations support the hypothesis of a link between the hemostatic system in the periphery and the malignant tumor disease even when the tumor is an intracerebral metastasis and the affected patient currently is free of a systemically active tumor. The results of this study support the hypothesis that the concerted action of coagulation factors and their receptors within the metastasis tissue itself and the systemic coagulation system could control the malignant behavior of tumor disease and make larger prospective trials mandatory.

Myelin-phagocytosing macrophages in isolated sciatic and optic nerves reveal a unique reactive phenotype.

Macrophages are key effectors in demyelinating diseases of the central and peripheral nervous system by phagocytosing myelin and releasing immunoregulatory mediators. Here, we report on a distinct, a priori anti-inflammatory reaction of macrophages phagocytosing myelin upon contact with damaged nerve tissue. Macrophages rapidly invaded peripheral (sciatic) and central (optic) nerve tissues in vitro, readily incorporated myelin and expressed high levels of phagocytosis-associated molecules (e.g., Fc and scavenger receptors). In contrast, factors involved in antigen presentation (MHC class-II, CD80, CD86) revealed only a restricted expression. In parallel, a highly ordered appearance of cytokines and chemokines was detected. IL-10, IL-6, CCL22, and CXCL1 were immediately but transiently induced, whereas CCL2, CCL11, and TGFbeta revealed more persisting levels. Such a profile would attract neutrophils, monocytes/macrophages, and Th2 cells as well as bias for a Th2-supporting environment. Importantly, proinflammatory/Th1-supporting factors, such as TNFalpha, IL-12p70, CCL3, and CCL5, were not induced. Still the simultaneous presence of TGFbeta and IL-6 could assist Th17 development, further depending on yet not present IL-23. The release pattern was clearly distinct from reactive phenotypes induced in isolated macrophages and microglia upon treatment with IL-4, IL-13, bacterial lipopolysaccharide, IFNgamma, or purified myelin. Nerve-exposed macrophages thus commit to a unique functional orientation.

Microglia.

Microglia--the macrophage equivalent of the CNS--safeguards and supports neuronal functions. Threats to the CNS homeostasis can trigger a rapid transformation of these cells from a normally "resting" into alerted and "activated" states. Microglia primarily serves the tissue defence and protection when participating in mechanisms of innate and adaptive immunity. On the contrary, excessive acute or chronic microglial activation can provoke severe neuronal and glial damage by carrying or fuelling destructive cascades. Several factors and conditions have already been identified that maintain the resting phenotype or organize and control the activation process. Cells are thereby able to recognize a dangerous signal as well as to sense functional disturbance. Microglial activation is also proving a much more variable and adaptive process than previously noticed. Aiming at microglia as a therapeutic target, research may focus on intracellular pathways that are probably common to activation scenarios as triggered by various receptor systems. Certain signalling elements may have key roles in the cytosolic integration of sensory inputs and a conversion into programs of executive performance. As the integrative aspect of microglial activation becomes illuminated hope builds up also on strategies for selective interference with harmful outcomes in favour of the--phylogenetically approved--beneficial potential of these fascinating cells.

The microglia-activating potential of thrombin: the protease is not involved in the induction of proinflammatory cytokines and chemokines.

The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase-activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia (brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non-proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous alpha- and gamma-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact alpha- and gamma-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease.