Chronic Oral Inoculation of Porphyromonas gingivalis and Treponema denticola Induce Different Brain Pathologies in a Mouse Model of Alzheimer Disease.

Periodontitis is a chronic inflammatory disease driven by dysbiosis in subgingival microbial communities leading to increased abundance of a limited number of pathobionts, including Porphyromonas gingivalis and Treponema denticola. Oral health, particularly periodontitis, is a modifiable risk factor for Alzheimer disease (AD) pathogenesis, with components of both these bacteria identified in postmortem brains of persons with AD. Repeated oral inoculation of mice with P. gingivalis results in brain infiltration of bacterial products, increased inflammation, and induction of AD-like biomarkers. P. gingivalis displays synergistic virulence with T. denticola during periodontitis. The aim of the current study was to determine the ability of P. gingivalis and T. denticola, grown in physiologically relevant conditions, individually and in combination, to induce AD-like pathology following chronic oral inoculation of female mice over 12 weeks. P. gingivalis alone significantly increased all 7 brain pathologies examined: neuronal damage, activation of astrocytes and microglia, expression of inflammatory cytokines interleukin 1ß (IL-1ß) and interleukin 6 and production of amyloid-ß plaques and hyperphosphorylated tau, in the hippocampus, cortex and midbrain, compared to control mice. T. denticola alone significantly increased neuronal damage, activation of astrocytes and microglia, and expression of IL-1ß, in the hippocampus, cortex and midbrain, compared to control mice. Coinoculation of P. gingivalis with T. denticola significantly increased activation of astrocytes and microglia in the hippocampus, cortex and midbrain, and increased production of hyperphosphorylated tau and IL-1ß in the hippocampus only. The host brain response elicited by oral coinoculation was less than that elicited by each bacterium, suggesting coinoculation was less pathogenic.

Omp31 of Brucella Inhibits NF-κB p65 Signaling Pathway by Inducing Autophagy in BV-2 Microglia.

Neurobrucellosis is a serious central nervous system (CNS) inflammatory disorder caused by Brucella, and outer membrane protein-31 (Omp31) plays an important role in Brucella infection. This study aims to determine whether Omp31 can induce autophagy in BV-2 microglia. Another goal of the study is to further examine the effect of autophagy on the nuclear transcription factor κB (NF-κB) p65 signaling pathway. We observed that Omp31 stimulated autophagy by increasing microtubule-associated protein 1 light chain 3B (LC3B-II) levels and inducing autophagosome formation at 6 h and 12 h. Concomitantly, Omp31 induced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression in a time-dependent manner but reduced the expression of TNF-α at 6 h. We utilized Omp31 with or without rapamycin or 3-methyladenine (3-MA) to treat BV-2 microglia, and it demonstrated further that Omp31 induced autophagy by promoting LC3B-II, Beclin-1 proteins expression and inhibiting the p62 protein levels. Furthermore, we explored the effects of autophagy on the NF-κB p65 pathway through western blot analysis, RT-qPCR assay, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The data suggest that Omp31 as well as rapamycin, the autophagy inducer, can decrease TNF-α levels through the inhibition of the NF-κB p65 signaling pathway. Taken together, Omp31 can function as a catalyst in both autophagy induction and NF-κB p65 signal inhibition. Furthermore, Omp31-induced autophagy may inhibit the expression of TNF-α by negatively regulating NF-κB p65 signaling pathway.

miR­4792 regulates inflammatory responses in Cryptococcus neoformans­infected microglia.

Investigating the factors that influence the inflammatory response of microglial cells is crucial for understanding the pathogenesis of cryptococcal meningitis (CM). MicroRNAs (miRNAs/miRs) play an important role in inducing host defenses and activating the immune response during microbial infection; however, the regulatory mechanisms of miRNAs in cryptococcal meningitis remain poorly defined. In a previous study, the authors assessed the miRNA profiles of THP­1 (human acute monocytic leukemia cells) cells following Cryptococcus neoformans (C. neoformans) infection. In the present study, it was found that miR­4792 expression was downregulated in BV2 cells infected with C. neoformans, whilst that of its target gene, epidermal growth factor receptor (EGFR), was upregulated. Infected cells in which miR­4792 was overexpressed exhibited a decreased EGFR transcript expression, reduced mitogen­activated protein kinase (MAPK) signaling and a decreased secretion of inflammatory cytokines. In addition, following antifungal treatment in patients with cryptococcal meningitis, the levels of miR­4792 in the cerebrospinal fluid significantly increased, whilst the expression of EGFR significantly decreased. In addition, receiver operator characteristic analysis revealed miR­4792 (AUCROC=0.75) and EGFR (AUCROC=0.79) as potential diagnostic markers in patients with cryptococcal meningitis.

Affinity of Mycobacterium tuberculosis strains for M059K microglial cells after migration through A549 alveolar epithelium.

Tuberculosis (TB) remains a major threat worldwide while central nervous system TB (CNS-TB) is one of the most severe forms of extrapulmonary TB. CNS-TB develops as a secondary infection during the hematogenous spread of Mycobacterium tuberculosis (M. tuberculosis) from the lungs to the CNS. Factors influencing the dissemination of the bacilli to the CNS have not been studied extensively. This study evaluated the transmigration ability through the alveolar epithelium and adhesion and invasion capacity of glial cells of M. tuberculosis strains of varying drug susceptibility and genotype profiles using an in vitro co-culture model. A549 alveolar epithelial cells and M059K glial cells were co-cultured in a Transwell plate with A549 cells cultured in the upper chamber and M059K glial cells in the lower chamber. A549 epithelial cells were infected with F15/LAM4/KZN (susceptible, MDR, XDR), Beijing (susceptible, XDR), F11 (susceptible), F28 (MDR), and H37Rv strains of M. tuberculosis. The transmigration of an A549 monolayer and subsequent adhesion and invasion rates of M059K cells were established. The susceptible and XDR variants of the F15/LAM4/KZN strain transmigrate the alveolar epithelial cell monolayer more efficiently than the MDR variant. The Beijing-XDR variant showed a high transmigration rate, while the susceptible variant showed no transmigration ability. Similar to the MDR F15/LAM4/KZN, the F28 and F11 strains showed a low dissemination ability. The bacteria were still capable to adhere to M059K glial cells after passage through the A549 cells. We conclude that M. tuberculosis isolates that passed through a monolayer of A549 alveolar epithelium by transcellular migration can still adhere to M059K glial cells. There is no genetic link between resistance and transmigration.

Oral P. gingivalis impairs gut permeability and mediates immune responses associated with neurodegeneration in LRRK2 R1441G mice.

BACKGROUND: The R1441G mutation in the leucine-rich repeat kinase 2 (LRRK2) gene results in late-onset Parkinson's disease (PD). Peripheral inflammation and gut microbiota are closely associated with the pathogenesis of PD. Chronic periodontitis is a common type of peripheral inflammation, which is associated with PD. Porphyromonas gingivalis (Pg), the most common bacterium causing chronic periodontitis, can cause alteration of gut microbiota. It is not known whether Pg-induced dysbiosis plays a role in the pathophysiology of PD. METHODS: In this study, live Pg were orally administrated to animals, three times a week for 1 month. Pg-derived lipopolysaccharide (LPS) was used to stimulate mononuclear cells in vitro. The effects of oral Pg administration on the gut and brain were evaluated through behaviors, morphology, and cytokine expression. RESULTS: Dopaminergic neurons in the substantia nigra were reduced, and activated microglial cells were increased in R1441G mice given oral Pg. In addition, an increase in mRNA expression of tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß) as well as protein level of α-synuclein together with a decrease in zonula occludens-1 (Zo-1) was detected in the colon in Pg-treated R1441G mice. Furthermore, serum interleukin-17A (IL-17A) and brain IL-17 receptor A (IL-17RA) were increased in Pg-treated R1441G mice. CONCLUSIONS: These findings suggest that oral Pg-induced inflammation may play an important role in the pathophysiology of LRRK2-associated PD.

CARD9+ microglia promote antifungal immunity via IL-1ß- and CXCL1-mediated neutrophil recruitment.

The C-type lectin receptor-Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clearance. In the present study we investigated host and pathogen factors that promote protective neutrophil recruitment during invasion of the CNS by Candida albicans. The cytokine IL-1ß served an essential function in CNS antifungal immunity by driving production of the chemokine CXCL1, which recruited neutrophils expressing the chemokine receptor CXCR2. Neutrophil-recruiting production of IL-1ß and CXCL1 was induced in microglia by the fungus-secreted toxin Candidalysin, in a manner dependent on the kinase p38 and the transcription factor c-Fos. Notably, microglia relied on CARD9 for production of IL-1ß, via both transcriptional regulation of Il1b and inflammasome activation, and of CXCL1 in the fungus-infected CNS. Microglia-specific Card9 deletion impaired the production of IL-1ß and CXCL1 and neutrophil recruitment, and increased fungal proliferation in the CNS. Thus, an intricate network of host-pathogen interactions promotes antifungal immunity in the CNS; this is impaired in human deficiency in CARD9, which leads to fungal disease of the CNS.

Microglia and amyloid precursor protein coordinate control of transient Candida cerebritis with memory deficits.

Bloodborne infections with Candida albicans are an increasingly recognized complication of modern medicine. Here, we present a mouse model of low-grade candidemia to determine the effect of disseminated infection on cerebral function and relevant immune determinants. We show that intravenous injection of 25,000 C. albicans cells causes a highly localized cerebritis marked by the accumulation of activated microglial and astroglial cells around yeast aggregates, forming fungal-induced glial granulomas. Amyloid precursor protein accumulates within the periphery of these granulomas, while cleaved amyloid beta (Aß) peptides accumulate around the yeast cells. CNS-localized C. albicans further activate the transcription factor NF-κB and induce production of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF), and Aß peptides enhance both phagocytic and antifungal activity from BV-2 cells. Mice infected with C. albicans display mild memory impairment that resolves with fungal clearance. Our results warrant additional studies to understand the effect of chronic cerebritis on cognitive and immune function.

Sex- and region-specific differences in microglia phenotype and characterization of the peripheral immune response following early-life infection in neonatal male and female rats.

Early-life infection has been shown to have profound effects on the brain and behavior across the lifespan, a phenomenon termed "early-life programming". Indeed, many neuropsychiatric disorders begin or have their origins early in life and have been linked to early-life immune activation (e.g. autism, ADHD, and schizophrenia). Furthermore, many of these disorders show a robust sex bias, with males having a higher risk of developing early-onset neurodevelopmental disorders. The concept of early-life programming is now well established, however, it is still unclear how such effects are initiated and then maintained across time to produce such a phenomenon. To begin to address this question, we examined changes in microglia, the immune cells of the brain, and peripheral immune cells in the hours immediately following early-life infection in male and female rats. We found that males showed a significant decrease in BDNF expression and females showed a significant increase in IL-6 expression in the cerebellum following E.coli infection on postnatal day 4; however, for most cytokines examined in the brain and in the periphery we were unable to identify any sex differences in the immune response, at least at the time points examined. Instead, neonatal infection with E.coli increased the expression of a number of cytokines in the brain of both males and females similarly including TNF-α, IL-1ß, and CD11b (a marker of microglia activation) in the hippocampus and, in the spleen, TNF-α and IL-1ß. We also found that protein levels of GRO-KC, MIP-1a, MCP1, IP-10, TNF-α, and IL-10 were elevated 8-hours postinfection, but this response was resolved by 24-hours. Lastly, we found that males have more thin microglia than females on P5, however, neonatal infection had no effect on any of the microglia morphologies we examined. These data show that sex differences in the acute immune response to neonatal infection are likely gene, region, and even time dependent. Future research should consider these factors in order to develop a comprehensive understanding of the immune response in males and females as these changes are likely the initiating agents that lead to the long-term, and often sex-specific, effects of early-life infection.

Neuroinvasive Listeria monocytogenes Infection Triggers IFN-Activation of Microglia and Upregulates Microglial miR-155.

MicroRNA (miR) miR-155 modulates microglial activation and polarization, but its role in activation of microglia during bacterial brain infection is unclear. We studied miR-155 expression in brains of C57BL/6 (B6.WT) mice infected i.p. with the neuro-invasive bacterial pathogen Listeria monocytogenes (L. monocytogenes). Infected mice were treated with ampicillin starting 2 days (d) post-infection (p.i.) and analyzed 3d, 7d, and 14d p.i. Virulent L. monocytogenes strains EGD and 10403s upregulated miR-155 in whole brain 7 d p.i. whereas infection with avirulent, non-neurotropic Δhly or ΔactA L. monocytogenes mutants did not. Similarly, infection with virulent but not mutated bacteria upregulated IFN-γ mRNA in the brain at 7 d p.i. Upregulation of miR-155 in microglia was confirmed by qPCR of flow cytometry-sorted CD45intCD11bpos brain cells. Subsequently, brain leukocyte influxes and gene expression in sorted microglia were compared in L. monocytogenes-infected B6.WT and B6.Cg-Mir155tm1.1Rsky/J (B6.miR-155-/-) mice. Brain influxes of Ly-6Chigh monocytes and upregulation of IFN-related genes in microglia were similar to B6.WT mice at 3 d p.i. In contrast, by d 7 p.i. expressions of microglial IFN-related genes, including markers of M1 polarization, were significantly lower in B6.miR-155-/- mice and by 14 d p.i., influxes of activated T-lymphocytes were markedly reduced. Notably, CD45highCD11bpos brain cells from B6.miR-155-/- mice isolated at 7 d p.i. expressed 2-fold fewer IFN-γ transcripts than did cells from B6.WT mice suggesting reduced IFN-γ stimulation contributed to dampened gene expression in B6.miR-155-/- microglia. Lastly, in vitro stimulation of 7 d p.i. brain cells with heat-killed L. monocytogenes induced greater production of TNF in B6.miR-155-/- microglia than in B6.WT microglia. Thus, miR-155 affects brain inflammation by multiple mechanisms during neuroinvasive L. monocytogenes infection. Peripheral miR-155 promotes brain inflammation through its required role in optimal development of IFN-γ-secreting lymphocytes that enter the brain and activate microglia. Microglial miR-155 promotes M1 polarization, and also inhibits inflammatory responses to stimulation by heat-killed L. monocytogenes, perhaps by targeting Tab2.

In vitro effects of commercial mouthwashes on several virulence traits of Candida albicans, viridans streptococci and Enterococcus faecalis colonizing the oral cavity.

Oral microbiota consists of hundreds of different species of bacteria, fungi, protozoa and archaea, important for oral health. Oral mycoses, mostly affecting mucosae, are mainly caused by the opportunistic pathogen Candida albicans. They become relevant in denture-wearers elderly people, in diabetic patients, and in immunocompromised individuals. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis and periodontitis, which affect even immune-competent individuals. An appropriate oral hygiene can avoid (or at least ameliorate) such pathologies: the regular and correct use of toothbrush, toothpaste and mouthwash helps prevent oral infections. Interestingly, little or no information is available on the effects (if any) of mouthwashes on the composition of oral microbiota in healthy individuals. Therefore, by means of in vitro models, we assessed the effects of alcohol-free commercial mouthwashes, with different composition (4 with chlorhexidine digluconate, 1 with fluoride, 1 with essential oils, 1 with cetylpyridinium chloride and 1 with triclosan), on several virulence traits of C. albicans, and a group of viridans streptococci, commonly colonizing the oral cavity. For the study here described, a reference strain of C. albicans and of streptococci isolates from pharyngeal swabs were used. Chlorhexidine digluconate- and cetylpyridinium chloride-containing mouthwashes were the most effective in impairing C. albicans capacity to adhere to both abiotic and biotic surfaces, to elicit proinflammatory cytokine secretion by oral epithelial cells and to escape intracellular killing by phagocytes. In addition, these same mouthwashes were effective in impairing biofilm formation by a group of viridans streptococci that, notoriously, cooperate with the cariogenic S. mutans, facilitating the establishment of biofilm by the latter. Differently, these mouthwashes were ineffective against other viridans streptococci that are natural competitors of S. mutans. Finally, by an in vitro model of mixed biofilm, we showed that mouthwashes-treated S. salivarius overall failed to impair C. albicans capacity to form a biofilm. In conclusion, the results described here suggest that chlorhexidine- and cetylpyridinium-containing mouthwashes may be effective in regulating microbial homeostasis of the oral cavity, by providing a positive balance for oral health. On the other side, chlorhexidine has several side effects that must be considered when prescribing mouthwashes containing this molecule.

Microglial immune response is impaired against the neurotropic fungus Lomentospora prolificans.

Lomentospora (Scedosporium) prolificans is an opportunistic pathogen capable of causing invasive infections in immunocompromised patients. The fungus is able to disseminate via the bloodstream finally arriving at the central nervous system producing neurological symptoms and, in many cases, patient death. In this context, microglial cells, which are the resident immune cells in the central nervous system, may play an important role in these infections. However, this aspect of anti-L. prolificans immunity has been poorly researched to date. Thus, the interactions and activity of microglial cells against L. prolificans were analysed, and the results show that there was a remarkable impairment in their performance regarding phagocytosis, the development of oxidative burst, and in the production of pro-inflammatory cytokines, compared with macrophages. Interestingly, L. prolificans displays great growth also when challenged with immune cells, even when inside them. We also proved that microglial phagocytosis of the fungus is highly dependent on mannose receptor and especially on dectin-1. Taken together, these data provide evidence for an impaired microglial response against L. prolificans and contribute to understanding the pathobiology of its neurotropism.

Mycobacterium marinum Infection in Zebrafish and Microglia Imitates the Early Stage of Tuberculous Meningitis.

Mycobacterium tuberculosis (M. tuberculosis) invading and activating microglia causes the most serious subtypes of tuberculosis called tubercular meningitis. However, the developmental process of tubercular meningitis, especially the early phase, is poorly understood due to lacking well-established and well-accepted visible models in vitro and in vivo. Here, consistent with one recent report, we found Mycobacterium marinum (M. marinum) invade the zebrafish brain and subsequently cause granuloma-like structures. We further showed that M. marinum, which shares similar characteristics with M. tuberculosis, can invade microglia and replicate in microglia, which subsequently promote the secretion of pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α. M. marinum infection in microglia can also promote autophagy, which conversely limits the replication of M. marinum. Thus, pharmacological activation of autophagy by rapamycin could prevent M. marinum replication. Our study provides in vivo and in vitro models to study underlying pathogenic mechanisms of tubercular meningitis by using M. marinum. Our results also showed that activation of autophagy could be a meaningful way to prevent tubercular meningitis.

The microbiota influences cell death and microglial colonization in the perinatal mouse brain.

The mammalian fetus develops in a largely sterile environment, and direct exposure to a complex microbiota does not occur until birth. We took advantage of this to examine the effect of the microbiota on brain development during the first few days of life. The expression of anti- and pro-inflammatory cytokines, developmental cell death, and microglial colonization in the brain were compared between newborn conventionally colonized mice and mice born in sterile, germ-free (GF) conditions. Expression of the pro-inflammatory cytokines interleukin 1ß and tumor necrosis factor α was markedly suppressed in GF newborns. GF mice also had altered cell death, with some regions exhibiting higher rates (paraventricular nucleus of the hypothalamus and the CA1 oriens layer of the hippocampus) and other regions exhibiting no change or lower rates (arcuate nucleus of the hypothalamus) of cell death. Microglial labeling was elevated in GF mice, due to an increase in both microglial cell size and number. The changes in cytokine expression, cell death and microglial labeling were evident on the day of birth, but were absent on embryonic day 18.5, approximately one-half day prior to expected delivery. Taken together, our results suggest that direct exposure to the microbiota at birth influences key neurodevelopmental events and does so within hours. These findings may help to explain some of the behavioral and neurochemical alterations previously seen in adult GF mice.

Infection of microglia with Porphyromonas gingivalis promotes cell migration and an inflammatory response through the gingipain-mediated activation of protease-activated receptor-2 in mice.

Despite a clear correlation between periodontitis and cognitive decline in Alzheimer's disease, the precise mechanism underlying the relationship remains unclear. The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Rgp and Kgp are important in the bacterial mediated host cell responses and the subsequent intracellular signaling in infected cells. In the present study, we attempted to clarify the potential effects of Rgp and Kgp on the cellular activation of brain-resident microglia. We provide the first evidence that Rgp and Kgp cooperatively contribute to the P. gingivalis-induced cell migration and expression of proinflammatory mediators through the activation of protease-activated receptor 2. The subsequent activation of phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathways contributes to cell migration and inflammatory response of microglia.

Activation of the TLR2-mediated downstream signaling pathways NF-κB and MAPK is responsible for B7-H3-augmented inflammatory response during S. pneumoniae infection.

It has been reported that B7-H3, a costimulatory protein, participates in the development and progression of experimental pneumococcal meningitis by amplifying the TLR2-mediated inflammatory response. This study attempted to clarify the pathway(s) of TLR2 signaling involved in B7-H3-augmented inflammatory response during S. pneumoniae infection. Murine microglial cell line N9 cells and primary murine microglial cells were infected with S. pneumoniae alone or in combination with B7-H3. Although B7-H3 stimulation failed to further enhance S. pneumoniae-upregulated mRNA and protein expression of TLR2, it strongly augmented S. pneumoniae-induced phosphorylation of NF-κB p65, MAPK p38, and ERK1/2 in both N9 cells and primary microglial cells. Notably, B7-H3 itself did not activate NF-κB p65, MAPK p38, and ERK1/2. Furthermore, deactivation of NF-κB p65, MAPK p38, and ERK1/2 with their specific inhibitors significantly attenuated B7-H3-amplified proinflammatory cytokine and chemokine release from S. pneumoniae-infected microglial cells. Importantly, blockage of NF-κB p65, MAPK p38, or ERK1/2 in vivo substantially diminished B7-H3-augmented TNF-α levels in the brain of S. pneumoniae-infected mice. These results indicate that the activation of both NF-κB and MAPKs is predominantly responsible for B7-H3-augmented inflammatory response during S. pneumoniae infection.

Cronobacter sakazakii clinical isolates overcome host barriers and evade the immune response.

Cronobacter sakazakii is the most frequently clinically isolated species of the Cronobacter genus. However the virulence factors of C. sakazakii including their ability to overcome host barriers remains poorly studied. In this study, ten clinical isolates of C. sakazakii were assessed for their ability to invade and translocate through human colonic carcinoma epithelial cells (Caco-2) and human brain microvascular endothelial cells (HBMEC). Their ability to avoid phagocytosis in human macrophages U937 and human brain microglial cells was investigated. Additionally, they were tested for serum sensitivity and the presence of the Cronobacter plasminogen activation gene (cpa) gene, which is reported to confer serum resistance. Our data showed that the clinical C. sakazakii strains invaded and translocated through Caco-2 and HBMEC cell lines and some strains showed significantly higher levels of invasion and translocation. Moreover, C. sakazakii was able to persist and even multiply in phagocytic macrophage and microglial cells. All strains, except one, were able to withstand human serum exposure, the single serum sensitive strain was also the only one which did not encode for the cpa gene. These results demonstrate that C. sakazakii clinical isolates are able to overcome host barriers and evade the host immune response indicating their capacity to cause diseases such as necrotizing enterocolitis (NEC) and meningitis. Our data showed for the first time the ability of C. sakazakii clinical isolates to survive and multiply within human microglial cells. Additionally, it was shown that C. sakazakii clinical strains have the capacity to translocate through the Caco-2 and HBMEC cell lines paracellularly.

TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis.

BACKGROUND: Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. METHODS: We generated neuron-specific TNF-deficient (NsTNF(-/-)) mice and compared outcomes of disease against TNF(f/f) control and global TNF(-/-) mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracerebral M. tuberculosis infection of TNF(-/-) mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF(-/-) mice were resistant to infection and presented with a phenotype similar to that in TNF(f/f) control mice. Impaired immunity in TNF(-/-) mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. CONCLUSION: Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB.

Systemic inflammation and microglial activation: systematic review of animal experiments.

BACKGROUND: Animal studies show that peripheral inflammatory stimuli may activate microglial cells in the brain implicating an important role for microglia in sepsis-associated delirium. We systematically reviewed animal experiments related to the effects of systemic inflammation on the microglial and inflammatory response in the brain. METHODS: We searched PubMed between January 1, 1950 and December 1, 2013 and Embase between January 1, 1988 and December 1, 2013 for animal studies on the influence of peripheral inflammatory stimuli on microglia and the brain. Identified studies were systematically scored on methodological quality. Two investigators extracted independently data on animal species, gender, age, and genetic background; number of animals; infectious stimulus; microglial cells; and other inflammatory parameters in the brain, including methods, time points after inoculation, and brain regions. RESULTS: Fifty-one studies were identified of which the majority was performed in mice (n = 30) or in rats (n = 19). Lipopolysaccharide (LPS) (dose ranging between 0.33 and 200 mg/kg) was used as a peripheral infectious stimulus in 39 studies (76 %), and live or heat-killed pathogens were used in 12 studies (24 %). Information about animal characteristics such as species, strain, sex, age, and weight were defined in 41 studies (80 %), and complete methods of the disease model were described in 35 studies (68 %). Studies were also heterogeneous with respect to methods used to assess microglial activation; markers used mostly were the ionized calcium binding adaptor molecule-1 (Iba-1), cluster of differentiation 68 (CD68), and CD11b. After LPS challenge microglial activation was seen 6 h after challenge and remained present for at least 3 days. Live Escherichia coli resulted in microglial activation after 2 days, and heat-killed bacteria after 2 weeks. Concomitant with microglial response, inflammatory parameters in the brain were reviewed in 23 of 51 studies (45 %). Microglial activation was associated with an increase in Toll-like receptor (TLR-2 and TLR-4), tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1ß) messenger ribonucleic acid (mRNA) expression or protein levels. INTERPRETATION: Animal experiments robustly showed that peripheral inflammatory stimuli cause microglial activation. We observed distinct differences in microglial activation between systemic stimulation with (supranatural doses) LPS and live or heat-killed bacteria.

Inflammatory mediator release from primary rhesus microglia in response to Borrelia burgdorferi results from the activation of several receptors and pathways.

BACKGROUND: In previous studies, neurons were documented to undergo apoptosis in the presence of microglia and live Borrelia burgdorferi, but not with either agent alone. Microscopy showed that several Toll-like receptors (TLRs) were upregulated in microglia upon B. burgdorferi exposure. It was hypothesized that the inflammatory milieu generated by microglia in the presence of B. burgdorferi results in neuronal apoptosis and that this inflammation was likely generated through TLR pathways. METHODS: In this study, we explored the role of several TLR and nucleotide-binding oligomerization domain containing 2 (NOD2)-dependent pathways in inducing inflammation in the presence of B. burgdorferi, using ribonucleic acid interference (RNAi) and/or inhibitors, in primary non-human primate (NHP) microglia. We also used several inhibitors for key mitogen-activated protein kinase (MAPK) pathways to determine the role of downstream pathways in inflammatory mediator release. RESULTS: The results show that the TLR2 pathway plays a predominant role in inducing inflammation, as inhibition of TLR2 with either small interfering RNA (siRNA) or inhibitor, in the presence of B. burgdorferi, significantly downregulated interleukin 6 (IL-6), chemokine (C-X-C) motif ligand 8 (CXCL8), chemokine (C-C) motif ligand 2 (CCL2), and tumor necrosis factor (TNF) production. This was followed by TLR5, the silencing of which significantly downregulated IL-6 and TNF. The role of TLR4 was inconclusive as a TLR4-specific inhibitor and TLR4 siRNA had opposing effects in the presence of B. burgdorferi. Silencing of NOD2 by siRNA in the presence of B. burgdorferi significantly upregulated IL-6, CCL2, and TNF. Downstream signaling involved the adaptor molecule myeloid differentiation primary response 88 (MyD88), as expected, as well as the MAPK pathways, with extracellular signal-regulated kinase (ERK) being predominant, followed by Jun N-terminal kinase (JNK) and p38 pathways. CONCLUSIONS: Several receptors and pathways, with both positive and negative effects, mediate inflammation of primary microglia in response to B. burgdorferi, resulting in a complex, tightly regulated immune network.

Retinal photoreceptor expresses toll-like receptors (TLRs) and elicits innate responses following TLR ligand and bacterial challenge.

Toll-like receptors (TLRs) play an important role in host defense against microbial pathogens. Our previous studies have shown that TLRs are expressed on various retinal cells (Microglia and Müller glia) and orchestrate retinal innate responses in bacterial endophthalmitis. In this study, we used a well-characterized mouse cone photoreceptor cell line (661W); and demonstrated that these cells express all known TLRs. Although the stimulation of 661W cells with TLR ligands (Pam3Cys, PolyI:C, LPS, Flagellin, Poly DT, and ODN) did not alter TLR expression, downstream TLR-signaling pathways (NF-κB, p38, and ERK) are activated. Moreover, TLR-activated 661W cells secreted significant amounts of inflammatory mediators (IL-6, IL-1ß, MIP-2, and KC) in their culture supernatant, as assessed by ELISA. A similar trend was observed in 661W cells challenged with live bacteria (Staphylococcus aureus). Interestingly, the neutralization of TLR2, a major receptor for S. aureus recognition, did not significantly attenuate bacterial-induced inflammatory mediators, suggesting the existence of TLR2-independent mechanisms in photoreceptor cells. Together, these results indicate that photoreceptors constitutively express functional TLRs and possess the ability to initiate innate responses following pathogen challenge, implicating their role in retinal innate immunity.