Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice

A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium ( S.  Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S.  Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1^+/+ and Nramp1^−/− mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S.  Typhimurium to systemic sites. We observed that Nramp1^+/+ mice showed lower numbers of S.  Typhimurium in the caecum compared with Nramp1^−/− mice at all times analysed. Acute inflammation was much more pronounced in Nramp1^+/+ mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-γ, TNF-α and MIP-1α and a massive infiltration of neutrophils and macrophages, compared with Nramp1^−/− animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S.  Typhimurium infection in Nramp1^+/+ mice.     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

MyD88 and IFN-alphabeta differentially control maturation of bystander but not Salmonella-associated dendritic cells or CD11cintCD11b+ cells during infection

The interface between dendritic cells (DCs) and T cells is critical to elicit effective immunity against pathogens. The maturation state of DCs determines the quality of the interaction and governs the type of response. DCs can be matured directly through activating Toll-like receptors (TLRs) or indirectly by cytokines. We explore the role of the TLR adaptor MyD88 on DC maturation during Salmonella infection. Using Salmonella expressing GFP, we also examine the phenotype and function of bacteria-associated DCs matured in the absence of bacteria-mediated TLR signalling. MyD88 was required for upregulation of CD80 on DCs during infection, whereas CD86 and CD40 were upregulated independently of MyD88, although requiring a higher bacterial burden in the MLN. MyD88-independent upregulation was mediated by IFN-αβ produced during infection. In infected MyD88^−/−IFN-αβR^−/− mice, which lack most bacteria-driven TLR signalling, indirect DC maturation was abolished. In contrast, DCs containing Salmonella upregulated co-stimulatory molecules independently of MyD88 and IFN-αβ, revealing a pathway of phenotypic maturation active in infected DCs. However, despite high co-stimulatory molecule expression, Salmonella -containing DCs from MyD88^−/− or MyD88^−/−IFN-αβR^−/− mice had a compromised capacity to activate T cells. Thus, bacterial stimulation of TLRs influences DC function at multiple levels that modulates their capacity to direct antibacterial immunity.     

Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection

Host resistance against Salmonella enterica serovar Typhimurium ( S . Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S . Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c⁺CD103^-) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c⁺CD103^- DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-α) than Nramp1−/−, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium.     

Toll-like receptor 4 is involved in neuroprotection afforded by ischemic preconditioning

It has been demonstrated that a short ischemic event (ischemic preconditioning, IPC) results in a subsequent resistance to severe ischemia (ischemic tolerance, IT). We have recently demonstrated the role of innate immunity and in particular of toll-like receptor (TLR) 4 in brain ischemia. Several evidences suggest that TLR4 might also be involved in IT. Therefore, we have now used an in vivo model of IPC to investigate whether TLR4 is involved in IT. A 6-min temporary bilateral common carotid arteries occlusion was used for focal IPC and it was performed on TLR4-deficient mice (C57BL/10ScNJ) and animals that express TLR4 normally (C57BL/10ScSn). To assess the ability of IPC to induce IT, permanent middle cerebral artery occlusion was performed 48 h after IPC. Stroke outcome was evaluated by determination of infarct volume and assessment of neurological scores. IPC caused neuroprotection as shown by a reduction in infarct volume and better outcome in mice expressing TLR4 normally. TLR4-deficient mice showed less IPC-induced neuroprotection than wild-type animals. Western blot analysis of tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) showed an up-regulation in the expression of these proteins in both substrains of mice measured 18, 24 and 48 h after IPC, being higher in mice with TLR4. Similarly, nuclear factor-kappa B (NF-κB) activation was observed 18, 24 and 48 h after IPC, being more intense in TLR4-expressing mice. These data demonstrate that TLR4 signalling is involved in brain tolerance as shown by the difference in the percentage of neuroprotection produced by IPC between ScSn and ScNJ (60% vs. 18%). The higher expression of TNF-α, iNOS and cyclooxygenase-2 and NF-κB activation in mice expressing TLR4 is likely to participate in this endogenous neuroprotective effect.     

A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.     

Tumor necrosis factor-alpha (TNF-alpha) regulates Toll-like receptor 2 (TLR2) expression in microglia

Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus ( S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus . In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-α (TNF-α) enhances TLR2 expression in microglia, whereas interleukin-1β has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-α, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-α-induced TLR2 expression. Similar results were observed with the IKK-2 and IκB-α inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-α was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-α knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-α knockout mice. Collectively, these results indicate that in response to S. aureus , TNF-α acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway.     

Therapeutic effectiveness of orally administered transgenic low-alkaloid tobacco expressing human interleukin-10 in a mouse model of colitis

Inflammatory bowel disease (IBD) represents a spectrum of diseases in which inflammation leads to acute and chronic gut injury. It is a growing health issue for which no cure exists. The pathogenesis is multifactorial with links to infectious and environmental events that trigger disease in genetically predisposed individuals. Treatment of the two major forms of IBD, Crohn's disease and ulcerative colitis, involves the reduction of inflammation with toxic immunosuppressive drugs or blocking of the pro-inflammatory effects of tumour necrosis factor-α (TNF-α) with antibodies. Here, we show that the oral administration of transgenic low-alkaloid tobacco expressing the contra-inflammatory cytokine human interleukin-10 (hIL-10) reduces the severity of colitis by down-regulating TNF-α expression directly at the sites of inflammation in IBD-susceptible IL-10^−/– mice. hIL-10 expressed in plants is biologically active and displays resistance to gastrointestinal degradation. Dietary supplementation with plant tissue delivering up to 9 µg of hIL-10 daily for 4 weeks was well tolerated by treated mice. Gut histology was significantly improved relative to controls ( P =  0.002), and was correlated with a decrease in small bowel TNF-α mRNA levels and an increase in IL-2 and IL-1β mRNA levels. Transgenic plants expressing IL-10 to directly attenuate TNF-α expression at sites of inflammation in the gut may become a useful new approach in the luminal therapy of IBD.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Cytoskeletal Changes in the Brains of Mice Lacking Calcineurin Aα

Abstract: Hyperphosphorylated τ, the major component of the paired helical filaments of Alzheimer's disease, was found to accumulate in the brains of mice in which the calcineurin Aα gene was disrupted [calcineurin Aα knockout (CNAα^−/−)]. The hyperphosphorylation involved several sites on τ, especially the Ser³⁹⁶ and/or Ser⁴⁰⁴ recognized by the PHF-1 monoclonal antibody. The increase in phosphorylated τ content occurred primarily in the mossy fibers of the CNAα^−/− hippocampus, which contained the highest level of calcineurin in brains of wild-type mice. The CNAα^−/− mossy fibers also contained less neurofilament protein than normal, although the overall level of neurofilament phosphorylation was unchanged. In the electron microscope, the mossy fibers of CNAα^−/− mice exhibited abnormalities in their cytoskeleton and a lower neurofilament/microtubule ratio than those of wild-type animals. These findings indicate that hyperphosphorylated τ can accumulate in vivo as a result of reduced calcineurin activity and is accompanied by cytoskeletal changes that are likely to have functional consequences on the affected neurons. The CNAα^−/− mice were found in a separate study to have deficits in learning and memory that may result in part from the cytoskeletal changes in the hippocampus.     

Effect of tumor necrosis factor-α on intracellular Staphylococcus aureus in vascular endothelial cells

Although tumor necrosis factor-α (TNF-α) is an important host factor against intracellular bacteria, little is known about the effect of TNF-α on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-α influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-α, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10⁶ CFU well⁻¹) into HUVECs for 1 h. TNF-α (1 ng mL⁻¹) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-α plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-α preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-α helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-α preferentially induces apoptosis of endothelial cells infected by S. aureus .     

Interferon-γ, interleukin-1 and tumour necrosis factor-α synthesis during experimental murine staphylococcal infection

Abstract Several exotoxins of Staphylococcus aureus were shown to modulate the host immune system by stimulation of monokine release. BALB/c mice infected intravenously (i.v.) with live cells if S. aureus , strain Cowan 1, had a detectable serum level of TNF-α at 3, 4 and 5 h after injection. When S. epidermidis (strain F3380, clinical isolate) was used to infect mice, the level of TNF-α was lower (the detection limit of the cytotoxicity assay with WEHI cells was 40 pg ml⁻). Kinetics of TNF synthesis was different from that observed in experimental infections caused by Gram-negative bacteria. Similarly to TNF-α, IL-1α appears in a measureable level at 3 h after i.v. injection of bacteria. The highest serum level of IFN-γ was observed 12 h after infection with both S. aureus and S. epidermidis . A quantity ten times more of S. epedermidis than of S. aureus cells was required to induce similar levels of TNF-α and IFN-γ administered in vivo in four daily doses followed by infection of S. aureus resulted in increased elimination of bacteria from the spleen, liver and peritoneal cavity of mice.     

Nitric oxide production: a mechanism of Chlamydia trachomatis inhibition in interferon-γ-treated RAW264.7 cells

Abstract IFN-γ and/or LPS induced nitrite production and inhibition of Chlamydia trachomatis (CT) replication in the murine macrophage cell line, RAW264.7. Linear regression analysis demonstrated a strong correlation between nitrite production and inhibition of CT replication (correlation coefficients: −0.93, P < 0.001). l -NMMA specifically inhibited nitrite production and restored CT replication (55–71%). Inducible nitric oxide synthase (iNOS) mRNA was analyzed by Northern and dot blot hybridization with an iNOS cDNA probe. A strong correlation between iNOS mRNA expression and inhibition of CT replication also was observed (correlation coefficient: −0.97, P < 0.05). Furthermore, anti-TNF-α antibody, which completely neutralized biological activity of the secreted TNF-α, neither inhibited nitrite production nor restored CT replication in the LPS- and/or IFN-γ-treated RAW264.7 cells. In mouse peritoneal macrophages treated with IFN-γ, both l -NMMA and anti-TNF-α antibody inhibited nitrite production and restored CT replication. However, l -NMMA and the antibody had no effect upon nitrite production and CT inhibition in LPS-treated peritoneal macrophages. These data indicate that NO production is one mechanism for inhibition of CT replication in IFN-γ-activated murine macrophages.     

Behavioral characterization of mice lacking the neurite outgrowth inhibitor Nogo-A

The membrane protein Nogo-A inhibits neurite outgrowth and regeneration in the injured central nervous system, primarily because of its expression in oligodendrocytes. Hence, deletion of Nogo-A enhances regeneration following spinal cord injury. Yet, the effects of Nogo-A deletion on general behavior and cognition have not been explored. The possibility of potential novel functions of Nogo-A beyond growth inhibition is strongly suggested by the presence of subpopulations of neurons also expressing Nogo-A – not only during development but also in adulthood. We evaluated here Nogo-A ^−/− mice in a series of general basic behavioral assays as well as functional analyses related to brain regions with notable expression levels of Nogo-A. The SHIRPA protocol did not show any major basic behavioral changes in Nogo-A ^−/− mice. Anxiety-related behavior, pain sensitivity, startle reactivity, spatial learning, and associative learning also appeared indistinguishable between Nogo-A ^−/− and control Nogo-A^+/+ mice. However, motor co-ordination and balance were enhanced in Nogo-A ^−/− mice. Spontaneous locomotor activity was also elevated in Nogo-A ^−/− mice, but this was specifically observed in the dark (active) phase of the circadian cycle. Enhanced locomotor reaction to systemic amphetamine in Nogo-A ^−/− mice further pointed to an altered dopaminergic tone in these mice. The present study is the first behavioral characterization of mice lacking Nogo-A and provides significant insights into the potential behavioral relevance of Nogo-A in the modulation of dopaminergic and motor functions.     

Genetic Basis of Vi Antigen Expression in Salmonella paratyphi C

Analysis of hybrids formed in a cross between a Salmonella paratyphi C Hfr and an S. typhimurium recipient indicated that the structural genetic determinants of the S. paratyphi C Vi antigen are located closely adjacent to the mel determinant, between this marker and purA. A similar location was indicated for the structural genetic determinants of the S. typhi Vi antigen (the viaB locus) by the results of a mating in which a hybrid S. typhimurium Hfr bearing the S. typhi viaB determinants was used to transfer these genes to an S. typhimurium recipient. Mating experiments with a Vi-antigen-expressing S. typhi Hfr and an S. typhimurium hybrid recipient expressing the Vi antigen of S. paratyphi C yielded no recombinants in which loss of Vi antigen expression occurred, indicating that the chromosomal locus occupied by the genetic determinants of the S. paratyphi C Vi antigen is the same one at which, in S. typhi, the viaB genes reside. Introduction of a mutant S. typhi viaA gene into an S. typhimurium hybrid expressing the Vi antigen, as the consequence of prior receipt of the S. paratyphi C viaB determinants, resulted in that hybrid's loss of Vi antigen expression, demonstrating that the viaA determinant plays a role in Vi antigen expression in S. paratyphi C, as well as in S. typhi. Although the percentages of coinheritance of the viaB and mel determinants in the mating experiments suggested that their linkage is sufficiently close to allow cotransduction by P22, attempts to accomplish this with lysates prepared on S. typhimurium hybrids expressing either S. typhi or S. paratyphi C viaB determinants were not successful.     

Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii.

Certain strains of the genus Citrobacter exhibit a variable expression of the Vi surface antigen that appears to involve a special mechanism for regulation of gene expression. Two nonlinked chromosomal loci, viaA and viaB, are known to determine nonvariable Vi antigen expression in strains of Salmonella. To confirm the presence of analogous loci in Citrobacter and to ascertain whether either of them is involved in variable Vi antigen expression in this organism, donor strains were constructed from Citrobacter freundii WR7004 and used to transfer their Vi antigen-determining genes to ViaA- and ViaB- Salmonella typhi recipient strains. Vi antigen expression in C. freundii was found to be controlled by loci analogous to the Salmonella via genes. S. typhi recipients of the C. freundii viaA+ genes were restored to the full, continuous expression of the Vi antigen normally seen in S. typhi. Thus, the C. freundii viaA genes appeared to play no role in the variable expression of the Vi antigen. In contrast, S. typhi recipients of the C. freundii viaB+ genes exhibited the rapid, reversible alternation between full Vi antigen expression and markedly reduced Vi antigen expression that was seen to occur in the C. freundii parent. The C. freundii viaB locus was thus identified as the one whose genes are regulated so as to produce variable Vi antigen expression. Genes determining another C. freundii surface antigen, the synthesis of which is not affected by the mechanism regulating Vi expression, were coinherited with the C. freundii viaB+ genes. An invertible, insertion sequence element located within the C. freundii viaB locus is proposed to account for the regulation of variable Vi antigen expression.     

Engagement of Penicillium marneffei conidia with multiple pattern recognition receptors on human monocytes

P. marneffei is a thermal dimorphic fungus which causes penicilliosis, an opportunistic infection in immunocompromised patients in South and Southeast Asia. Little is known about the innate immune response to P. marneffei infection. Therefore, the initial response of macrophages to P. marneffei conidia was evaluated by us. Adhesion between monocytes from healthy humans and fungal conidia was examined and found to be specifically inhibited by MAbs against PRR, such as MR, (TLR)1, TLR2, TLR4, TLR6, CD14, CD11a, CD11b, and CD18. To study the consequences of these interactions, cytokines were also examined by ELISA. Binding of P. marneffei conidia to monocytes was significantly inhibited, in a dose-dependent manner, by MAbs against MR, TLR1, TLR2, TLR4, TLR6, CD14, CD11b and CD18. When monocytes were co-cultured with the conidia, there was an increase in the amount of surface CD40 and CD86 expression, together with TNF-α and IL-1β production, compared to unstimulated controls. In assays containing anti-TLR4 or anti-CD14 antibody, reduction in the amount of TNF-α released by monocytes stimulated with P. marneffei conidia was detected. In addition, it was found that production of TNF-α and IL-1β from adherent peripheral blood monocytes was partially impaired when heat-inactivated autologous serum, in place of untreated autologous serum, was added to the assay. These results demonstrate that various PRR on human monocytes participate in the initial recognition of P. marneffei conidia, and the engagement of PRR could partly initiate proinflammatory cytokine production.