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.

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.

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

BACKGROUND: Palmitoylethanolamide (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. METHODS: In 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. RESULTS: Stimulation 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-1ß and IL-6 in homogenates of spleen and cerebellum in mice treated with PEA. CONCLUSIONS: Our 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.

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.

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

BACKGROUND: Toll-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. METHODS: We studied the ability of microglial cells to increase the phagocytosis of cryptococci after stimulation with agonists of TLR1/2, TLR3, TLR4 and TLR9. RESULTS: Stimulation 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. CONCLUSION: Our 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.

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.

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/mm(2); 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.

Higher mortality and impaired elimination of bacteria in aged mice after intracerebral infection with E. coli are associated with an age-related decline of microglia and macrophage functions.

Incidence and mortality of bacterial meningitis are strongly increased in aged compared to younger adults demanding new strategies to improve prevention and therapy of bacterial central nervous system (CNS) infections the elderly. Here, we established a geriatric mouse model for an intracerebral E. coli infection which reflects the clinical situation in aged patients: After intracerebral challenge with E. coli K1, aged mice showed a higher mortality, a faster development of clinical symptoms, and a more pronounced weight loss. Elimination of bacteria and systemic inflammatory response were impaired in aged mice, however, the number of infiltrating leukocytes and microglial cells in the CNS of aged and young mice did not differ substantially. In vitro, primary microglial cells and peritoneal macrophages from aged mice phagocytosed less E. coli and released less NO and cyto-/chemokines compared to cells from young mice both without activation and after stimulation by agonists of TLR 2, 4, and 9. Our results suggest that the age-related decline of microglia and macrophage functions plays an essential role for the higher susceptibility of aged mice to intracerebral infections. Strategies to improve the phagocytic potential of aged microglial cells and macrophages appear promising for prevention and treatment of CNS infections in elderly patients.

Palmitoylethanolamide stimulates phagocytosis of Escherichia coli K1 and Streptococcus pneumoniae R6 by microglial cells.

The ability of microglial cells to phagocytose bacteria after stimulation with the endocannabinoid palmitoylethanolamide (PEA) was studied in vitro. PEA increased the phagocytosis of unencapsulated Streptococcus pneumoniae R6 and encapsulated Escherichia coli K1 by murine microglial cells significantly after 30 min of microglial stimulation. This suggested that stimulation of microglial cells by PEA can increase the resistance of the brain against CNS infections.

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

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 µg/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.

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.