miR-155 and miR-146a collectively regulate meningitic Escherichia coli infection-mediated neuroinflammatory responses.

BACKGROUND: Escherichia coli is the most common Gram-negative bacterium causing meningitis, and E. coli meningitis is associated with high mortality and morbidity throughout the world. Our previous study showed that E. coli can colonize the brain and cause neuroinflammation. Increasing evidence supports the involvement of miRNAs as key regulators of neuroinflammation. However, it is not clear whether these molecules participate in the regulation of meningitic E. coli-mediated neuroinflammation. METHODS: The levels of miR-155 and miR-146a, as well as their precursors, in E. coli-infected astrocytes were measured using quantitative real-time PCR (qPCR). Overexpression and knockdown studies of miR-155 and miR-146a were performed to observe the effects on bacterial loads, cytokines, chemokines, and NF-κB signaling pathways. Bioinformatics methods were utilized to predict the target genes, and these target genes were validated using qPCR, Western blotting, and luciferase reporter system. In vivo knockdown of miR-155 and miR-146a was carried out to observe the effects on bacterial loads, inflammatory genes, astrocyte activation, microglia activation, and survival in a mouse model. RESULTS: The levels of miR-155, miR-146a, and their precursors were significantly increased in astrocytes during E. coli infection. miR-155 and miR-146a were induced by the NF-κB-p65 signaling pathway upon infection. Overexpressing and inhibiting miR-155 and miR-146a in astrocytes did not affect the bacterial loads. Further, the in vitro overexpression of miR-155 and miR-146a suppressed the E. coli-induced inflammatory response, whereas the inhibition of miR-155 and miR-146a enhanced it. Mechanistically, miR-155 inhibited TAB2, and miR-146a targeted IRAK1 and TRAF6; therefore, they functioned collaboratively to modulate TLR-mediated NF-κB signaling. In addition, both miR-155 and miR-146a could regulate the EGFR-NF-κB signaling pathway. Finally, the in vivo suppression of E. coli-induced miR-155 and miR-146a further promoted the production of inflammatory cytokines, aggravated astrocyte and microglia activation, and decreased mouse survival time, without affecting the bacterial loads in the blood and brain. CONCLUSIONS: E. coli infection induced miR-155 and miR-146a, which collectively regulated bacteria-triggered neuroinflammatory responses through negative feedback regulation involving the TLR-mediated NF-κB and EGFR-NF-κB signaling pathways, thus protecting the central nervous system from further neuroinflammatory damage.

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.

Efficacy of Urtoxazumab (TMA-15 Humanized Monoclonal Antibody Specific for Shiga Toxin 2) Against Post-Diarrheal Neurological Sequelae Caused by Escherichia coli O157:H7 Infection in the Neonatal Gnotobiotic Piglet Model.

Enterohemorrhagic Escherichia coli (EHEC) is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited), a humanized monoclonal antibody against Shiga toxin (Stx) 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 × 108 colony-forming units of EHEC O157:H7 strain EDL933 (Stx1⁺, Stx2⁺) when 22-24 h old. At 24 h post-inoculation, piglets were intraperitoneally administered placebo or TMA-15 (0.3, 1.0 or 3.0 mg/kg body weight). Compared to placebo (n = 10), TMA-15 (n = 35) yielded a significantly greater probability of survival, length of survival, and weight gain (p <0.05). The efficacy of TMA-15 against brain lesions and death was 62.9% (p = 0.0004) and 71.4% (p = 0.0004), respectively. These results suggest that TMA-15 may potentially prevent or reduce vascular necrosis and infarction of the brain attributable to Stx2 in human patients acutely infected with EHEC. However, we do not infer that TMA-15 treatment will completely protect human patients infected with EHEC O157:H7 strains that produce both Stx1 and Stx2.

The effects of cytotoxic necrotizing factor 1 expression in the uptake of Escherichia coli K1 by macrophages and the onset of meningitis in newborn mice.

Macrophages are a permissive niche for E. coli K1 multiplication for which the interaction of the bacterial outer membrane protein A and its cognate receptor CD64 are critical. Using in vitro immunofluorescence and live microscopy with ex vivo macrophage cultures from RFP-Lifeact mice, we show that cytotoxic necrotizing factor 1 (CNF1) secreted by E. coli K1 sequesters cellular actin toward microspike formation, thereby limiting actin availability for OmpA-mediated bacterial invasion. Surprisingly, the observed effects of CNF1 occur despite the absence of 67-kDa laminin receptor in macrophages. Concomitantly, the CNF1 deletion mutant of E. coli K1 (Δcnf1) invades macrophages and the brains of newborn mice in greater numbers compared to wild-type. However, the Δcnf1 strain induces less severe pathology in the brain. These results suggest a novel role for CNF1 in limiting E. coli K1 entry into macrophages while exacerbating disease severity in the brains of newborn mice.

Recombinant OmpA protein fragments mediate interleukin-17 regulation to prevent Escherichia coli meningitis.

BACKGROUND: Neonates are at a higher risk for bacterial meningitis than children of other age groups. Although the mortality rates have decreased over the past few decades, neonatal meningitis is still a severe disease with high morbidity. For bacterial meningitis, antibiotic therapy is the primary choice for management. However, neurologic complications often cannot be averted; ∼40% of survivors exhibit neurological sequelae. Escherichia coli infection is the common cause of neonatal meningitis. Previously, we have demonstrated that the recombinant loop 1-3, loop 2-3, and loop 2-4 fragments of OmpA protein can protect mice from death after intracerebral E. coli infection. In this study, the protective effects of the recombinant OmpA protein fragments in E. coli intracerebral infections were investigated. METHODS: The effects of E. coli intracerebral infection on cytokine and chemokine expression were determined. We also used various recombinant fragments of the OmpA protein to investigate the effects of these recombinant OmpA protein fragments on cytokine and chemokine expression. RESULTS: In this study, we demonstrated that the expression of interleukin-17 and other cytokines, chemokines, inducible nitric oxide synthase, and cyclooxygenase-2 are involved in the inflammatory processes of intracerebral E. coli infection. We also demonstrated that specific recombinant OmpA protein fragments (L1-3, L2-3, L2-4, and L3) can regulate cytokine, chemokine, nitric oxide synthase, and cyclooxygenase-2 expression and, subsequently, protect mice from death caused by intracerebral infection of E. coli. CONCLUSION: This finding indicates the potential for developing a new therapeutic approach to improve the prognosis of bacterial meningitis.

Recent advances in adherence and invasion of pathogenic Escherichia coli.

PURPOSE OF REVIEW: Colonization of the host epithelia by pathogenic Escherichia coli is influenced by the ability of the bacteria to interact with host surfaces. Because the initial step of an E. coli infection is to adhere, invade, and persist within host cells, some strategies used by intestinal and extraintestinal E. coli to infect host cell are presented. RECENT FINDINGS: This review highlights recent progress understanding how extraintestinal pathogenic E. coli strains express specific adhesins or invasins that allow colonization of the urinary tract or the meninges, while intestinal E. coli strains are able to colonize different regions of the intestinal tract using other specialized adhesins or invasins. Finally, evaluation of different diets and environmental conditions regulating the colonization of these pathogens is discussed. SUMMARY: Discovery of new interactions between pathogenic E. coli and the host epithelial cells unravels the need for more mechanistic studies that can provide new clues regarding how to combat these infections.

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.

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.

Involvement of IbeA in meningitic Escherichia coli K1-induced polymorphonuclear leukocyte transmigration across brain endothelial cells.

Transmigration of neutrophil [polymorphonuclear neutrophil (PMN)] across the blood-brain barrier (BBB) is a critical event in the pathogenesis of bacterial meningitis. We have shown that IbeA is able to induce meningitic Escherichia coli invasion of brain microvascular endothelial cells (BMECs), which constitutes the BBB. In this report, we provide evidence that IbeA and its receptor, vimentin, play a key role in E. coli-induced PMN transmigration across BMEC. In vitro and in vivo studies indicated that the ibeA-deletion mutant ZD1 was significantly less active in stimulating PMN transmigration than the parent strain E44. ZD1 was fully complemented by the ibeA gene and its product. E. coli-induced PMN transmigration was markedly inhibited by withaferin A, a dual inhibitor of vimentin and proteasome. These cellular effects were significantly stimulated and blocked by overexpression of vimentin and its head domain deletion mutant in human BMEC, respectively. Our studies further demonstrated that IbeA-induced PMN migration was blocked by bortezomib, a proteasomal inhibitor and correlated with upregulation of endothelial ICAM-1 and CD44 expression through proteasomal regulation of NFκB activity. Taken together, our data suggested that IbeA and vimentin contribute to E. coli K1-stimulated PMN transendothelial migration that is correlated with upregulation of adhesion molecule expression at the BBB.

IL-10 administration reduces PGE-2 levels and promotes CR3-mediated clearance of Escherichia coli K1 by phagocytes in meningitis.

Ineffectiveness of antibiotics in treating neonatal Escherichia coli K1 meningitis and the emergence of antibiotic-resistant strains evidently warrants new prevention strategies. We observed that administration of interleukin (IL)-10 during high-grade bacteremia clears antibiotic-sensitive and -resistant E. coli from blood of infected mice. Micro-CT studies of brains from infected animals displayed gross morphological changes similar to those observed in infected human neonates. In mice, IL-10, but not antibiotic or anti-TNF antibody treatment prevented brain damage caused by E. coli. IL-10 administration elevated CR3 expression in neutrophils and macrophages of infected mice, whereas infected and untreated mice displayed increased expression of FcgammaRI and TLR2. Neutrophils or macrophages pretreated with IL-10 ex vivo exhibited a significantly greater microbicidal activity against E. coli compared with cells isolated from wild-type or IL-10-/- mice. The protective effect of IL-10 was abrogated when CR3 was knocked-down in vivo by siRNA. The increased expression of CR3 in phagocytes was caused by inhibition of prostaglandin E-2 (PGE-2) levels, which were significantly increased in neutrophils and macrophages upon E. coli infection. These findings describe a novel modality of IL-10-mediated E. coli clearance by diverting the entry of bacteria via CR3 and preventing PGE-2 formation in neonatal meningitis.

Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-kappaB activation.

Phagocytes are well-known effectors of the innate immune system to produce proinflammatory cytokines and chemokines such as tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-8 during infections. Here, we show that infection of monocytes with wild-type Escherichia coli K1, which causes meningitis in neonates, suppresses the production of cytokines and chemokines (TNF-alpha, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1beta, IL-1beta, and IL-8). In contrast, infection of monocytes with a mutant E. coli, which lacks outer membrane protein A (OmpA- E. coli) resulted in robust production of cytokines and chemokines. Wild-type E. coli K1 (OmpA+ E. coli) prevented the phosphorylation and its degradation of inhibitor of kappaB, thereby blocking the translocation of nuclear factor (NF)-kappaB to the nucleus. OmpA+ E. coli-infected cells, subsequently subjected to lipopolysaccharide challenge, were crippled severely in their ability to activate NF-kappaB to induce cytokine/chemokine production. Selective inhibitors of the extracellular signal-regulated kinase (ERK) 1/2 pathway and p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase, significantly reduced the activation of NF-kappaB and the production of cytokines and chemokines induced by OmpA- E. coli, indicating a role for these kinases in the NF-kappaB/cytokine pathway. It is interesting that the phosphorylation of ERK 1/2 and p38 MAPK was notably reduced in monocytes infected with OmpA+ E. coli when compared with monocytes infected with OmpA- E. coli, suggesting that the modulation of upstream events common for NF-kappaB and MAPKs by the bacterium is possible. The ability of OmpA+ E. coli K1 to inhibit the macrophage response temporarily may enable bacterial survival and growth within the host for the onset of meningitis by E. coli K1.

Differential regulation of Toll-like receptor mRNAs in experimental murine central nervous system infections.

Toll-like receptors (TLR) play a key role in the recognition of microbial components. We investigated the differential regulation of TLR mRNA expression in bacterial and viral mouse models of central nervous system infection. Streptococcus pneumoniae meningitis led to an enhanced expression of TLR2, TLR4 and TLR9 mRNA. In Escherichia coli meningitis, TLR2, TLR4 and TLR7 mRNA expression was increased and Herpes simplex encephalitis caused a rise of TLR4 mRNA. In organotypic hippocampal cultures treatment with S. pneumoniae R6 led to increased expression of TLR2 and TLR3 mRNA. Our data provide evidence that regulation of TLR mRNA is not fully specific for the molecular patterns of the infectious pathogen. The TLR mRNA regulation observed probably represents a combination of specific response to the causative pathogen and non-specific activation of the innate immune system.

N(omega) -nitro-L-arginine methyl ester (L-NAME) attenuates the acute inflammatory responses and brain injury during the early phase of experimental Escherichia coli meningitis in the newborn piglet.

We evaluated the anti-inflammatory and neuroprotective effect of nonselective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), in experimental bacterial meningitis in the newborn piglet. Meningitis was induced by intracisternal injection of 10(8) colony forming units of Escherichia coli. L-NAME 10 mg kg(-1) was given intravenously 30 min before induction of meningitis. L-NAME significantly attenuated the increase in intracranial pressure and decrease in cerebrospinal fluid glucose concentration observed in the meningitis group. Systemic and cerebral perfusion pressure were even higher compared to the control and meningitis groups. However, the meningitis-induced increase in tumor necrosis factor-alpha level, leukocyte numbers and lactate level in the cerebrospinal fluid was not significantly attenuated with L-NAME administration. Reduced cerebral cortical cell membrane Na+, K+ -ATPase activity and increased lipid peroxidation products, indicative of meningitis-induced brain cell membrane dysfunction, were significantly improved with L-NAME treatment. Decreased brain glucose and ATP levels were also significantly improved with L-NAME treatment. These findings suggest that L-NAME was effective in attenuating the acute inflammatory responses and brain injury in neonatal bacterial meningitis.