The Salmonella LysR family regulator, RipR, activates the SPI-13 encoded itaconate degradation cluster

The Salmonella LysR Family Regulator RipR Activates the SPI-13-Encoded Itaconate Degradation Cluster

Infection and Immunity ◽

10.1128/iai.00303-20 ◽

2020 ◽

Vol 88 (10) ◽

Author(s):

Steven J. Hersch ◽

William Wiley Navarre

Keyword(s):

Isocitrate Lyase ◽

Inducible Promoter ◽

Specific Response ◽

Neighboring Gene ◽

Content Type ◽

Bactericidal Effects ◽

Mouse Macrophages ◽

Monocyte Cell

ABSTRACT Itaconate is a dicarboxylic acid that inhibits the isocitrate lyase enzyme of the bacterial glyoxylate shunt. Activated macrophages have been shown to produce itaconate, suggesting that these immune cells may employ this metabolite as a weapon against invading bacteria. Here, we demonstrate that in vitro, itaconate can exhibit bactericidal effects under acidic conditions similar to the pH of a macrophage phagosome. In parallel, successful pathogens, including Salmonella, have acquired a genetic operon encoding itaconate degradation proteins, which are induced heavily in macrophages. We characterized the regulation of this operon by the neighboring gene ripR in specific response to itaconate. Moreover, we developed an itaconate biosensor based on the operon promoter that can detect itaconate in a semiquantitative manner and, when combined with the ripR gene, is sufficient for itaconate-regulated expression in Escherichia coli. Using this biosensor with fluorescence microscopy, we observed bacteria responding to itaconate in the phagosomes of macrophages and provide additional evidence that gamma interferon stimulates macrophage itaconate synthesis and that J774 mouse macrophages produce substantially more itaconate than the human THP-1 monocyte cell line. In summary, we examined the role of itaconate as an antibacterial metabolite in mouse and human macrophages, characterized the regulation of Salmonella’s defense against it, and developed it as a convenient itaconate biosensor and inducible promoter system.

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Interaction of mouse splenocytes and macrophages with bacterial strains in vitro: the effect of age in the immune response

Beneficial Microbes ◽

10.3920/bm2015.0094 ◽

2016 ◽

Vol 7 (2) ◽

pp. 275-287 ◽

Cited By ~ 5

Author(s):

A.A. Van Beek ◽

J.A. Hoogerland ◽

C. Belzer ◽

P. De Vos ◽

W.M. De Vos ◽

...

Keyword(s):

Immune System ◽

Immune Cells ◽

Inflammatory Responses ◽

Interferon Γ ◽

Bacterial Strains ◽

Mouse Splenocytes ◽

Aged Mice ◽

Inflammatory Conditions ◽

Cellular Inflammatory Response

Probiotics influence the immune system, both at the local and systemic level. Recent findings suggest the relation between microbiota and the immune system alters with age. Our objective was to address direct effects of six bacterial strains on immune cells from young and aged mice: Lactobacillus plantarum WCFS1, Lactobacillus casei BL23, Lactococcus lactis MG1363, Bifidobacterium breve ATCC15700, Bifidobacterium infantis ATCC15697, and Akkermansia muciniphila ATCC BAA-835. We used splenocytes and naïve or interferon-γ-stimulated bone marrow-derived macrophages (BMDM) as responder populations. All tested bacterial strains induced phenotypic and cytokine responses in splenocytes and BMDM. Based on magnitude of the cellular inflammatory response and cytokine profiles, two subgroups of bacteria were identified, i.e. L. plantarum and L. casei versus B. breve, B. infantis, and A. muciniphila. The latter group of bacteria induced high levels of cytokines produced under inflammatory conditions, including tumour necrosis factor (TNF), interleukin (IL)-6 and IL-10. Responses to L. lactis showed features of both subgroups. In addition, we compared responses by splenocytes and BMDM derived from young mice to those of aged mice, and found that splenocytes and BMDM derived from aged mice had an increased IL-10 production and dysregulated IL-6 and TNF production compared to young immune cells. Overall, our study shows differential inflammatory responses to distinct bacterial strains, and profound age-dependent effects. These findings, moreover, support the view that immune environment importantly influences bacterial immune effects.

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Investigating the effects of novel heparan sulfate mimetic 'HS16-35' on immune cell migration in an in vitro model of the blood-CSF barrier.

10.26686/wgtn.17009102.v1 ◽

2021 ◽

Author(s):

◽

Maddie Griffiths

Keyword(s):

Multiple Sclerosis ◽

Central Nervous System ◽

Nervous System ◽

Heparan Sulfate ◽

Choroid Plexus ◽

Immune Cells ◽

Low Dose ◽

Interferon Γ ◽

The Central Nervous System

The central nervous system was traditionally considered an immune-privileged site, defined as being immunologically inactive. However, recent studies have elucidated that a number of immune cells traffic into and out of the brain in healthy humans to conduct routine immunosurveillance. A unique immunological interface, the choroid plexus, acts as a gatekeeper for the entry of these immune cells during homeostasis. Although the mechanisms are not well described, the choroid plexus also has the capacity to regulate the responses of migrating leukocytes during inflammation. Multiple sclerosis is a complex neuroinflammatory disease characterized by demyelination in the CNS. Autoreactive immune cells invade the central nervous system and orchestrate an attack against myelin sheathes, the insulation layer that protects neurons. The disease affects nearly 1 in 1,000 New Zealanders, and currently has no cure. The most successful treatments for multiple sclerosis target the initial stages by inhibiting the entry of these cells into the central nervous system, however these are often associated with severe side and life-threatening effects and cannot prevent the progression of the disease. Heparanase, the ubiquitously expresses heparan sulfate degrading enzyme has been thoroughly implicated in the disease processes of multiple sclerosis, and its animal model, EAE. Autoreactive lymphocytes exploit heparanase activity to degrade the extracellular matrix and destabilize the barriers that maintain the relative immune privileged status of the central nervous system. Exogenous heparan sulfate mimetics have previously been shown to ameliorate symptoms of EAE by interfering with heparanase activity. However, the commercialization and clinical translation of these inhibitors is currently inhibited by the complexity of their synthesis. ‘HS16-35’ is a novel heparan sulfate mimetic developed by the Ferrier Institute, comprised of a dendritic core with four heavily sulfated oligosaccharide arms. The synthesis of this compound is much shorter due to its smaller size; however, it has been shown to act similarly to native heparan sulfate molecules. We proposed that HS16-35 is protective in preventing the migration of autoreactive immune cells across the choroid plexus by inhibiting lymphocyte heparanase. To investigate the efficacy of HS16-35 in vitro, we first established an experimental transwell model of the choroid plexus. This model incorporated core components of the choroid plexus, including fenestrated capillaries, the stromal matrix and epithelial monolayer. We first showed that the model was capable of mimicking homeostatic trafficking across the choroid plexus epithelium, which formed a selective but permeable barrier. Then, we induced T-cell specific inflammatory migration using Concanavalin A or TH1-type cytokines. This migration was found to be interferon-γ dependent and could be mitigated with anti-interferon-γ treatment. Once this model was established, we next investigated whether HS16-35 was effective in inhibiting inflammatory migration across this structure. To adapt HS16-35 to an in vitro dose, we performed cell viability assays. This confirmed that the compound was mildly cytotoxic to epithelial choroid plexus cells but not murine splenocytes. Further experiments found that low-dose HS16-35 did not impact monolayer permeability. Transwell migration assays showed that low-dose HS16-35 was effective in reducing ConA and interferon-γ mediated inflammatory T-cell migration to a level comparable to homeostatic trafficking. Finally, we assessed cytokine profiles of leukocytes and epithelial choroid plexus cells treated with HS16 35 and found that HS16-35 reduced the expression of key cytokines involved in MS pathogenesis. In summary, the work described in this thesis shows how HS16-35 may be protective during EAE by suppressing the inflammatory response of autoreactive T-cells, in addition to regulating the infiltration of immune cells into the CNS through the choroid plexus. In a broader sense, these findings show that HS16 36 may be effective in treating MS by regulating, not inhibiting lymphocyte migration into the CNS, mitigating some of the severe side effects that other migration-inhibitors face.

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In vitro Killing of Entamoeba histolytica trophozoites by interferon-γ-activated mouse macrophages

Immunobiology ◽

10.1016/s0171-2985(88)80018-4 ◽

1988 ◽

Vol 176 (4-5) ◽

pp. 341-353 ◽

Cited By ~ 11

Author(s):

Esfandiar Ghadiriani ◽

Daniel T. Bout

Keyword(s):

Entamoeba Histolytica ◽

Interferon Γ ◽

Mouse Macrophages

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Interferon-γ Mediated Pathways And Mitogen Stimulated Proliferation During And After An Acute Infection

Pteridines ◽

10.1515/pteridines-2018-0005 ◽

2018 ◽

Vol 29 (1) ◽

pp. 70-79

Author(s):

Miriam Knoll ◽

Dietmar Fuchs ◽

Guenter Weiss ◽

Rosa Bellmann-Weiler ◽

Bojana Kovrlija ◽

...

Keyword(s):

Immune System ◽

Immune Cells ◽

Bacterial Infections ◽

Acute Infection ◽

Interferon Γ ◽

Immune System Activation ◽

System Activation ◽

Acute Bacterial Infections

AbstractBackground: Interferon-γ (IFN- γ) regulates the degradation of tryptophan to kynurenine via induction of indoleamine- 2,3-dioxygenase (IDO). Local tryptophan depletion and accumulation of toxic metabolites might impair the proliferative capacity of lymphocytes. The aim of this study was to assess the actual status of immune system activation of patients with bacterial infection in the acute phase and during convalescence in vivo and in vitro. Parameters of systemic immune system activation were evaluated for associations with proliferative responsiveness of immune cells, and compared with healthy controls. Methods: 24 patients with various acute bacterial infections were included in the group of acutely ill patients. Sixteen patients participated in a follow-up examination after convalescence. The control group consisted of 6 healthy people. To assess the status of immune system activation in vivo, inflammation parameters C-reactive protein and differential blood counts were determined. Neopterin concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Tryptophan and kynurenine measurements were performed with high pressure liquid chromatography (HPLC). Peripheral blood mononuclear cells (PBMCs) were isolated from the patients’ blood and stimulated with concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) in vitro proliferation rates were evaluated by ³H-thymidine incorporation and neopterin production and tryptophan degradation were determined in supernatants of mitogen stimulated PBMCs. Results: Patients with acute bacterial infections showed reduced tryptophan and elevated neopterin concentrations, which did not normalize after convalescence period. Higher plasma neopterin values and increased IDO-activity were associated with reduced proliferative responses in vitro after stimulation with PHA. Associations were observed during acute infection as well as convalescence. Conclusions: Results of this study show that increased immune system activation in vivo is associated with impaired proliferative responsiveness of immune cells in vitro in acute bacterial infections as well as during convalescence.

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Increased GILZ expression in transgenic mice up-regulates Th-2 lymphokines

Blood ◽

10.1182/blood-2005-05-2183 ◽

2006 ◽

Vol 107 (3) ◽

pp. 1039-1047 ◽

Cited By ~ 64

Author(s):

Lorenza Cannarile ◽

Francesca Fallarino ◽

Massimiliano Agostini ◽

Salvatore Cuzzocrea ◽

Emanuela Mazzon ◽

...

Keyword(s):

Transgenic Mice ◽

Leucine Zipper ◽

Cell Lineage ◽

Lymphocyte Activation ◽

Interferon Γ ◽

Interleukin 4 ◽

Delayed Type Hypersensitivity ◽

Specific Response ◽

Th 1

AbstractGILZ (glucocorticoid-induced leucine zipper), a gene induced by dexamethasone, is involved in control of T lymphocyte activation and apoptosis. In the present study, using Gilz transgenic mice (TG), which overexpress GILZ in the T-cell lineage, we demonstrate that Gilz is implicated in T helper-2 (Th-2) response development. After in vitro stimulation by CD3/CD28 antibodies, peripheral naive CD4+ T cells from TG mice secrete more Th-2 cytokines such as interleukin-4 (IL-4), IL-5, IL-13, and IL-10, and produce less Th-1 cytokines such as interferon-γ (IFN-γ) than wild-type mice (WT). CD4+ TG lymphocytes up-regulated Th-2 cytokine expression in the specific response to ovalbumin chicken egg (OVA) antigen immunization. Up-regulation correlated with increased expression of GATA-3 and signal transducer and activator of transcription 6 (Stat6), Th-2–specific transcription factors and decreased expression of T-bet, a transcription factor involved in Th-1 differentiation. Finally, in TG mice delayed-type hypersensitivity, a Th-1 response, was inhibited and bleomycin-induced pulmonary fibrosis, a Th-2 mediated disease, was more severe. These results indicate that Gilz contributes to CD4+ commitment toward a Th-2 phenotype and suggest this contribution may be another mechanism accounting for glucocorticoid immunomodulation.

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In Vitro Immuno-Modulatory Potentials of Purslane (Portulaca oleracea L.) Polysaccharides with a Chemical Selenylation

Foods ◽

10.3390/foods11010014 ◽

2021 ◽

Vol 11 (1) ◽

pp. 14

Author(s):

Ya-Ru Lin ◽

Qing-Yun Guan ◽

Ling-Yu Li ◽

Zhi-Mei Tang ◽

Qiang Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Immune Cells ◽

Immune Modulation ◽

Interferon Γ ◽

Portulaca Oleracea ◽

Edible Plant ◽

Murine Splenocytes ◽

Cd8 Cells ◽

Macrophage Phagocytosis

The soluble polysaccharides from a non-conventional and edible plant purslane (Portulaca oleracea L.), namely PSPO, were prepared by the water extraction and ethanol precipitation methods in this study. The obtained PSPO were selenylated using the Na2SeO3-HNO3 method to successfully prepare two selenylated products, namely SePSPO-1 and SePSPO-2, with different selenylation extents. The assay results confirmed that SePSPO-1 and SePSPO-2 had respective Se contents of 753.8 and 1325.1 mg/kg, while PSPO only contained Se element about 80.6 mg/kg. The results demonstrated that SePSPO-1 and SePSPO-2 had higher immune modulation than PSPO (p < 0.05), when using the two immune cells (murine splenocytes and RAW 264.7 macrophages) as two cell models. Specifically, SePSPO-1 and SePSPO-2 were more active than PSPO in the macrophages, resulting in higher cell proliferation, greater macrophage phagocytosis, and higher secretion of the immune-related three cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β. Meanwhile, SePSPO-1 and SePSPO-2 were more potent than PSPO in the concanavalin A- or lipopolysaccharide-stimulated splenocytes in cell proliferation, or more able than PSPO in the splenocytes to promote interferon-γ secretion but suppress IL-4 secretion, or more capable of enhancing the ratio of T-helper (CD4+) cells to T-cytotoxic (CD8+) cells for the T lymphocytes than PSPO. Overall, the higher selenylation extent of the selenylated PSPO mostly caused higher immune modulation in the model cells, while a higher polysaccharide dose consistently led to the greater regulation effect. Thus, it is concluded that the employed chemical selenylation could be used in the chemical modification of purslane or other plant polysaccharides, when aiming to endow the polysaccharides with higher immuno-modulatory effect on the two immune cells.

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LYMPHOCYTE PROLIFERATION IN VITRO INDUCED BY HAPTEN AUTOLOGOUS PROTEIN CONJUGATES

Journal of Experimental Medicine ◽

10.1084/jem.139.3.732 ◽

1974 ◽

Vol 139 (3) ◽

pp. 732-753 ◽

Cited By ~ 13

Author(s):

Bent Rubin ◽

Hans Wigzell

Keyword(s):

Lymph Node ◽

T Lymphocytes ◽

Dna Synthesis ◽

Present Article ◽

Immune Cells ◽

Guinea Pigs ◽

Differential Sensitivity ◽

Specific Response ◽

Lymph Node Cells

Immune lymph node cells from guinea pigs respond to soluble antigen in vitro by an increase in DNA synthesis. Optimal conditions for this proliferative response were studied in the present article. Under such conditions, immune cells showed increasing responses with increasing antigen concentration in vitro, the threshold dose of activation frequently being as low as 0.02 µg per culture. In contrast, normal lymph node cells (from FCA-stimulated animals) did only respond to antigen at very high doses (20 mg/culture), and immune cell dilution studies could be performed in normal cells without changing the kinetics of the antigen specific response of immune cells. Fractionation on anti-Ig columns indicated that purified, immune T lymphocytes were quite capable of proliferating in vitro upon antigen stimulation. However, our attempts to adsorb the proliferating cells onto chemically defined immunoadsorbants failed despite the fact that immune B cells (as measured by the rosette assay) were retained almost completely by such a procedure. Purified, immune T lymphocytes from guinea pigs immunized with different antigen concentrations in vivo and/or obtained at different times after immunization were tested for a differential sensitivity toward antigen-induced DNA synthesis in vitro. However, we were not able to demonstrate any regular increase in sensitivity to antigen in vitro, and if found, it seemed to be more dependent upon the number of antigen reactive cells in the population studied rather than upon differences in the average avidity of the receptors on the cells proliferating in vitro. The results in the present article are discussed in relation to current knowledge and hypotheses on T-lymphocyte receptors.

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Murine brain endothelial cells differently modulate interferon-γ and interleukin-17 production in vitro

Archives of Biological Sciences ◽

10.2298/abs0901029m ◽

2009 ◽

Vol 61 (1) ◽

pp. 29-36

Author(s):

Miljana Momcilovic ◽

D. Miljkovic ◽

Marija Mostarica-Stojkovic

Keyword(s):

Endothelial Cells ◽

Immune Cells ◽

Interferon Γ ◽

Interleukin 17 ◽

Brain Endothelial Cell ◽

Brain Endothelial Cells ◽

Cns Inflammation ◽

Lymph Node Cells ◽

Pro Inflammatory Cytokines

Brain endothelial cells (BEC) are the major constituents of the blood-brain barrier (BBB), the structure that controls entrance of immune cells into CNS parenchyma. Our aim was to investigate the influence of BEC on production of IL-17 and IFN-?-cytokines that are important for CNS inflammation. To that end, co-cultivations of the bEnd.3 brain endothelial cell line and lymph node cells (LNC) were performed, and gene expression and production of IL-17 and IFN-? were determined. It was found that bEnd.3 cells inhibited expression and production of IFN-?, but not of IL-17. Additionally, bEnd.3 cells also reduced production of the major IFN-?-promoting cytokine - IL-12 - in LNC. The observed variation in modulation of pro-inflammatory cytokines by BEC could be of importance for the understanding of CNS inflammation.

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Investigating the effects of novel heparan sulfate mimetic 'HS16-35' on immune cell migration in an in vitro model of the blood-CSF barrier.

10.26686/wgtn.17009102 ◽

2021 ◽

Author(s):

◽

Maddie Griffiths

Keyword(s):

Multiple Sclerosis ◽

Central Nervous System ◽

Nervous System ◽

Heparan Sulfate ◽

Choroid Plexus ◽

Immune Cells ◽

Low Dose ◽

Interferon Γ ◽

The Central Nervous System

The central nervous system was traditionally considered an immune-privileged site, defined as being immunologically inactive. However, recent studies have elucidated that a number of immune cells traffic into and out of the brain in healthy humans to conduct routine immunosurveillance. A unique immunological interface, the choroid plexus, acts as a gatekeeper for the entry of these immune cells during homeostasis. Although the mechanisms are not well described, the choroid plexus also has the capacity to regulate the responses of migrating leukocytes during inflammation. Multiple sclerosis is a complex neuroinflammatory disease characterized by demyelination in the CNS. Autoreactive immune cells invade the central nervous system and orchestrate an attack against myelin sheathes, the insulation layer that protects neurons. The disease affects nearly 1 in 1,000 New Zealanders, and currently has no cure. The most successful treatments for multiple sclerosis target the initial stages by inhibiting the entry of these cells into the central nervous system, however these are often associated with severe side and life-threatening effects and cannot prevent the progression of the disease. Heparanase, the ubiquitously expresses heparan sulfate degrading enzyme has been thoroughly implicated in the disease processes of multiple sclerosis, and its animal model, EAE. Autoreactive lymphocytes exploit heparanase activity to degrade the extracellular matrix and destabilize the barriers that maintain the relative immune privileged status of the central nervous system. Exogenous heparan sulfate mimetics have previously been shown to ameliorate symptoms of EAE by interfering with heparanase activity. However, the commercialization and clinical translation of these inhibitors is currently inhibited by the complexity of their synthesis. ‘HS16-35’ is a novel heparan sulfate mimetic developed by the Ferrier Institute, comprised of a dendritic core with four heavily sulfated oligosaccharide arms. The synthesis of this compound is much shorter due to its smaller size; however, it has been shown to act similarly to native heparan sulfate molecules. We proposed that HS16-35 is protective in preventing the migration of autoreactive immune cells across the choroid plexus by inhibiting lymphocyte heparanase. To investigate the efficacy of HS16-35 in vitro, we first established an experimental transwell model of the choroid plexus. This model incorporated core components of the choroid plexus, including fenestrated capillaries, the stromal matrix and epithelial monolayer. We first showed that the model was capable of mimicking homeostatic trafficking across the choroid plexus epithelium, which formed a selective but permeable barrier. Then, we induced T-cell specific inflammatory migration using Concanavalin A or TH1-type cytokines. This migration was found to be interferon-γ dependent and could be mitigated with anti-interferon-γ treatment. Once this model was established, we next investigated whether HS16-35 was effective in inhibiting inflammatory migration across this structure. To adapt HS16-35 to an in vitro dose, we performed cell viability assays. This confirmed that the compound was mildly cytotoxic to epithelial choroid plexus cells but not murine splenocytes. Further experiments found that low-dose HS16-35 did not impact monolayer permeability. Transwell migration assays showed that low-dose HS16-35 was effective in reducing ConA and interferon-γ mediated inflammatory T-cell migration to a level comparable to homeostatic trafficking. Finally, we assessed cytokine profiles of leukocytes and epithelial choroid plexus cells treated with HS16 35 and found that HS16-35 reduced the expression of key cytokines involved in MS pathogenesis. In summary, the work described in this thesis shows how HS16-35 may be protective during EAE by suppressing the inflammatory response of autoreactive T-cells, in addition to regulating the infiltration of immune cells into the CNS through the choroid plexus. In a broader sense, these findings show that HS16 36 may be effective in treating MS by regulating, not inhibiting lymphocyte migration into the CNS, mitigating some of the severe side effects that other migration-inhibitors face.

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