Sandra Redlich

Toll-like receptor stimulation increases phagocytosis of Cryptococcus neoformans by microglial cells

BackgroundToll-Like receptors (TLRs) belong to the family of pattern-recognition receptors with a crucial function of recognising pathogen-associated molecular patterns (PAMPs). Cryptococcal meningitis is a potentially fatal disease with a high mortality and risk of neurological sequelae.MethodsWe studied the ability of microglial cells to increase the phagocytosis of cryptococci after stimulation with agonists of TLR1/2, TLR3, TLR4 and TLR9.ResultsStimulation of murine microglial cells with these TLR agonists for 24 h increased the phagocytosis of encapsulated Cryptococcus neoformans. Stimulation increased the release of TNF-α, CXCL1 (KC), IL-6, IL-10 and MIP-2, which indicated the activation of microglial cells. Unstimulated and TLR agonist-stimulated MyD88-deficient cells showed a reduced ability to phagocytose cryptococci compared to their wild-type counterpart. Intracellular killing of cryptococci was also increased in TLR-stimulated cells compared to unstimulated microglial cells.ConclusionOur observation suggests that stimulation of microglial cells by TLR agonists can increase the resistance of the brain against CNS infections caused by Cryptococcus neoformans. This may be of interest when an immunocompromised patient is unable to eliminate Cryptococcus neoformans despite antifungal therapy.

Vitamin D Deficiency Reduces the Immune Response, Phagocytosis Rate, and Intracellular Killing Rate of Microglial Cells

ABSTRACT 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.

Palmitoylethanolamide stimulates phagocytosis of Escherichia coli K1 by macrophages and increases the resistance of mice against infections

BackgroundPalmitoylethanolamide (PEA), an endogenous lipid and a congener of anandamide, possesses a wide range of effects related to metabolic and cellular homeostasis including anti-inflammatory and neuroprotective properties.MethodsIn vitro, we studied the ability of macrophages to phagocytose Escherichia coli K1 after stimulation with increasing doses of PEA. In vivo, wild-type mice were treated with PEA intraperitoneally 12 hours and 30 minutes before infection. Meningoencephalitis or sepsis was induced by intracerebral or intraperitoneal infection with E. coli K1.ResultsStimulation of macrophages with PEA for 30 minutes increased the phagocytosis of E. coli K1 without inducing the release of TNFα or CXCL1. Intracellular killing of E. coli K1 was higher in PEA-stimulated than in unstimulated peritoneal macrophages and microglial cells. Pre-treatment with PEA significantly increased survival of mice challenged intracerebrally or intraperitoneally with E. coli K1. This effect was associated with a decreased production of CXCL1, IL-1β and IL-6 in homogenates of spleen and cerebellum in mice treated with PEA.ConclusionsOur observations suggest that these protective effects of PEA in mice can increase the resistance to bacterial infections without the hazard of collateral damage by excessive stimulation of phagocytes.

The nucleotide-binding oligomerization domain-containing-2 ligand muramyl dipeptide enhances phagocytosis and intracellular killing of Escherichia coli K1 by Toll-like receptor agonists in microglial cells

Increasing the phagocytic activity of microglia could improve the resistance of immunocompromised patients to CNS infections. We studied the microglial responses upon stimulation with the Nod2 ligand muramyl dipeptide (MDP) alone or in combination with a TLR1/2, 3 or 4 agonist. MDP caused a mild release of NO, but induced neither a significant release of pro-inflammatory cytokines nor an expression of molecules associated with professional antigen presentation. Using the Escherichia coli K1 model, microglial pre-stimulation with MDP enhanced bacterial phagocytosis which was strengthened on TLR-pre-stimulated cells. Dual pre-stimulation of Nod2 and TLR1/2 or 4 caused maximal phagocytosis and intracellular killing.

Multivalent choline dendrimers increase phagocytosis of streptococcus pneumoniae R6 by microglial cells

Background: Pneumococcal virulence factors common to all serotypes, such as choline-binding proteins (CBPs), are promising therapeutic targets in pneumococcal infections. We studied the effect of a choline dendrimer with maximized binding affinity/specificity for CBPs on microglia-mediated pneumococcal phagocytosis. Methods: Pneumoccocal cultures were exposed to dendrimers containing 8 choline end groups or amino groups as controls, either from the beginning of bacterial growth or at the late exponential phase. The effect of long/short co-incubation was assessed in terms of bacterial morphological changes and increase in bacterial uptake by primary microglial cultures. Results: Inhibiting CBPs by micromolar concentrations of a choline dendrimer caused the formation of long pneumococcal chains that were readily phagocytosed by microglia. Enhanced phagocytosis was dendrimer dose-dependent. Long bacteria-dendrimer co-incubation (14 h) resulted in a higher bacterial uptake than short co-incubation (2 h; p < 0.001). Conclusions: Multivalent dendrimers containing choline end groups are promising antimicrobial agents for the management of pneumococcal diseases.

Follistatin does not influence the course of Escherichia coli K1 sepsis in a mouse model.

ABSTRACT Follistatin (FS) is the binding protein of activin A and inhibits its actions. The activin/FS system participates in the fine tuning of the immune response, and concentrations of activin A and FS are elevated in serum of patients with sepsis. Intraperitoneal injection of FS markedly reduced mortality after lipopolysaccharide-induced inflammation in a mouse model. Here, we investigated whether FS also influences the disease course in a mouse model of sepsis induced by intraperitoneal injection of Escherichia coli K1, a gram-negative bacterium frequently causing septic bacterial infections. Intraperitoneal injection of 10 &mgr;g/mL FS 30 min before infection did not influence survival, weight, motor performance, or bacterial titers of the infected mice. Thus, we could not confirm the protective effect of FS observed during lipopolysaccharide-induced inflammation in our mouse model of E. coli sepsis. Although it is a promising therapeutic tool in chronic or acute inflammatory conditions not caused by virulent pathogens, FS does not seem to increase the resistance to bacterial infections.

Intrathecal Treatment with the Anti-Phosphorylcholine Monoclonal Antibody TEPC-15 Decreases Neuronal Damage in Experimental Pneumococcal Meningitis

Background: Neuronal injury in pneumococcal meningitis is a consequence of microglial activation and direct toxicity by bacterial products and systemic inflammation. Methods: The treatment effect of the TEPC-15 antibody recognizing teichoic and lipoteichoic acids was investigated in murine microglial cells and in a rabbit model of pneumococcal meningitis. Results: In vitro, the TEPC-15 antibody recognizing teichoic and lipoteichoic acids increased Streptococcus pneumoniae phagocytosis by murine microglial cells. In rabbit ceftriaxone-treated S. pneumoniae meningitis, intracisternal TEPC-15 reduced the density of apoptotic neurons in the hippocampal dentate gyrus (116 ± 70 vs. 221 ± 132/mm2; p = 0.03). Cerebrospinal fluid inflammatory parameters (protein, lactate, leukocytes, prostaglandins) were not reduced in TEPC-15-treated rabbits. Conclusion: Intracisternal treatment with the TEPC-15 antibody reduced neuronal damage probably by promoting rapid phagocytosis of bacterial products.

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.