Jörg Scheffel

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.

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.

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

Significance Microglia (brain macrophages) become rapidly activated in most neuropsychiatric disorders. A popular concept is that a single pathogenic stimulus, such as bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4), is sufficient to induce a reactive proinflammatory phenotype in microglia that exerts neurotoxicity. This concept is biologically risky, however. Here we provide evidence that chronic activation with either LPS or the leukocyte cytokine IFN-γ induces different reactive phenotypes in microglia of postnatal hippocampal tissue. Notably, these phenotypes only moderately alter diverse neuronal functions. In contrast, coactivation of TLR4 and IFN-γ receptors results in massive neural dysfunction and death. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ signaling from other host immune cells to induce neurodegeneration. 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-1β, 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.

Influence of methylene blue on microglia-induced inflammation and motor neuron degeneration in the SOD1(G93A) model for ALS.

Mutations in SOD1 cause hereditary variants of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous, with toxicity deriving also from glia. In particular, microglia contribute to disease progression. Methylene blue (MB) inhibits the effect of nitric oxide, which mediates microglial responses to injury. In vivo 2P-LSM imaging was performed in ALS-linked transgenic SOD1G93A mice to investigate the effect of MB on microglia-mediated inflammation in the spinal cord. Local superfusion of the lateral spinal cord with MB inhibited the microglial reaction directed at a laser-induced axon transection in control and SOD1G93A mice. In vitro, MB at high concentrations inhibited cytokine and chemokine release from microglia of control and advanced clinical SOD1G93A mice. Systemic MB-treatment of SOD1G93A mice at early preclinical stages significantly delayed disease onset and motor dysfunction. However, an increase of MB dose had no additional effect on disease progression; this was unexpected in view of the local anti-inflammatory effects. Furthermore, in vivo imaging of systemically MB-treated mice also showed no alterations of microglia activity in response to local lesions. Thus although systemic MB treatment had no effect on microgliosis, instead, its use revealed an important influence on motor neuron survival as indicated by an increased number of lumbar anterior horn neurons present at the time of disease onset. Thus, potentially beneficial effects of locally applied MB on inflammatory events contributing to disease progression could not be reproduced in SOD1G93A mice via systemic administration, whereas systemic MB application delayed disease onset via neuroprotection.

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. GLIA 2013;61:1331–1346

Fibronectin is elevated in the cerebrospinal fluid of patients suffering from bacterial meningitis and enhances inflammation caused by bacterial products in primary mouse microglial cell cultures.

Toll‐like receptors (TLR) play a key role in the recognition of pathogenic organisms. Fibronectin, an extracellular matrix protein, is considered a potent stimulator of the innate immune system through TLR4. In bacterial meningitis, several extracellular matrix proteins and bacterial compounds are elevated in the CSF. For this reason, we hypothesized that these molecules may jointly stimulate the innate immune system and increase neuronal damage in bacterial meningitis. Concentrations of fibronectin were elevated in the CSF of patients suffering from bacterial meningitis, but not in patients with multiple sclerosis, when compared with control patients without CSF abnormalities. In primary cultures of mouse microglial cells, co‐administration of fibronectin at concentrations occurring in the CSF in bacterial meningitis (10 μg/mL) with defined TLR agonists [lipopolysaccharide (TLR4), the synthetic lipopeptide tripalmytoyl‐cysteinyl‐seryl‐(lysyl)3‐lysine (TLR2) and single‐stranded unmethylated cytosine‐guanosine oligodesoxynucleotide (TLR9)] led to an additive release of nitric oxide and tumor necrosis factor‐alpha when compared with the release elicited by either compound alone. In conclusion, the inflammatory reaction to bacterial compounds can be aggravated by endogenous fibronectin at elevated levels during bacterial CNS infections. This additive or synergistic effect may contribute to neuronal damage during bacterial meningitis.

Fibronectin stimulates Escherichia coli phagocytosis by microglial cells.

Microglia express Toll‐like receptors (TLRs) that recognize invading pathogens as well as endogenous proteins such as fibronectin under nonphysiological conditions. Here, we demonstrated that fibronectin stimulates murine microglia in culture in a dose‐dependent manner: microglial cells secreted proinflammatory cytokines and chemokines and increased phagocytosis of Escherichia coli DH5α and E. coli K1 strains. Low levels of fibronectin exerted a synergistic effect on the release of proinflammatory compounds by microglia co‐stimulated with agonists for TLR1/2 (Pam3CSK4) or TLR9 (CpG DNA), but not in combination with the TLR4 agonist lipopolysaccharide (LPS). Phagocytosis of bacterial strains was moderately enhanced when microglia was co‐stimulated with high concentrations of fibronectin and one pathogen‐derived TLR agonist. In conclusion, fibronectin increased proinflammatory and phagocytotic functions in microglia and partially synergized with microbial TLR agonists.

Regulation of the tyrosine phosphorylation of Phospholipid Scramblase 1 in mast cells that are stimulated through the high-affinity IgE receptor.

Engagement of high-affinity immunoglobulin E receptors (FcεRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3-kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FcεRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRγ chain. FcεRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FcεRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FcεRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.

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 Brutons 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. GLIA 2015;63:1083–1099

Reduced release of nitric oxide and different cytokines/chemokines by aged microglial cells upon activation of Toll-like receptors 2 and 4

Objectives: The incidence of bacterial infections of the central nervous system (CNS) in adults increases with age, and the clinical outcome in elderly individuals is worse than in young persons. Death in the acute phase of the infection and neurologic or neuropsychologic deficits are common complications, and better therapies are needed. Since microglial cells play a key role for the defence of the brain against bacterial infections, we investigated age-related functional changes of these cells in vitro. Methods: Primary microglial cultures were obtained from brains of C57BL/6-N mice aged 2 months (young) and 18 months (aged). For activation, cells cultured in 96-well-plates were treated with agonists of Toll-like receptor (TLR) 1/2 [Tripalmitoyl-S-glycerylcysteine (Pam3CSK4); 0.1 μg/ml] and TLR 4 [endotoxin (LPS) from Escherichia coli serotype 026:B6; 0.01 μg/ml] for 24 h in the presence of interferon-gamma (IFN-gamma; 100 U/ml). Non-activated control cells were treated with IFN-gamma only. Young and aged microglial cells were compared regarding their release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and CXCL1 (KC) into the cell culture supernatant. Results: Upon stimulation with agonists of TLR 2 and 4, aged microglial cells released significantly less NO and cytokines/ chemokines than young microglial cells: nitrite upon Pam3CSK4 treatment: 6.97 (5.96/8.28) vs. 18.40 (8.78/20.89) μM, p = 0.008; TNF-alpha upon LPS treatment: 284.10 (262.40/300.50) vs. 681.20 (468.50/1024.00) pg/ml, p = 0.002; IL-6 upon Pam3CSK4 treatment: 85.70 (17.86/144.30) vs. 924.90 (82.00/937.90) pg/ml, p = 0.04; KC upon LPS treatment: 15.63 (15.63/24.23) vs. 75.00 (44.10/117.40) pg/ml, p = 0.007 [representative results, concentrations in the cell culture supernatant indicated as median (25./75. percentile), comparison aged vs. young by Mann–Whitney-U-test, n = 5–8]. Conclusion: Our in-vitro data show a reduced release of NO and inflammatory cytokines/chemokines by aged microglial cells upon activation with bacterial TLR agonists. The age-related decline of microglial functions might contribute to the higher susceptibility of elderly individuals to bacterial CNS infections. Strategies to improve the functions of aged microglial cells appear promising for prevention and treatment of CNS infections in elderly patients.