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1.
PLoS One ; 10(6): e0128794, 2015.
Article in English | MEDLINE | ID: mdl-26053018

ABSTRACT

BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.


Subject(s)
Carrageenan/administration & dosage , Carrageenan/therapeutic use , Influenza A virus/drug effects , Orthomyxoviridae Infections/drug therapy , Zanamivir/administration & dosage , Zanamivir/therapeutic use , Administration, Intranasal , Animals , Antiviral Agents/therapeutic use , Carrageenan/pharmacology , Disease Models, Animal , Dogs , Humans , Influenza A Virus, H7N7 Subtype/drug effects , Inhibitory Concentration 50 , Madin Darby Canine Kidney Cells , Mice , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/virology , Treatment Outcome , Zanamivir/pharmacology
2.
PLoS One ; 8(3): e60476, 2013.
Article in English | MEDLINE | ID: mdl-23544144

ABSTRACT

In this study we investigated the role of Bruton's tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk (-/-) BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk (-/-) BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk (-/-) BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk (-/-) mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk (-/-) mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.


Subject(s)
Listeria monocytogenes/physiology , Listeriosis/enzymology , Listeriosis/microbiology , Macrophages/enzymology , Macrophages/microbiology , Protein-Tyrosine Kinases/metabolism , Agammaglobulinaemia Tyrosine Kinase , Animals , Bone Marrow Cells/pathology , Cytokines/biosynthesis , Disease Susceptibility , Enzyme Activation/drug effects , Immunoblotting , Lipopeptides/pharmacology , Listeria monocytogenes/drug effects , Listeriosis/pathology , MAP Kinase Signaling System/drug effects , Macrophages/drug effects , Mice , Phagocytosis/drug effects , Phagosomes/drug effects , Phagosomes/microbiology , Protein-Tyrosine Kinases/deficiency
3.
Int Arch Allergy Immunol ; 161(1): 44-52, 2013.
Article in English | MEDLINE | ID: mdl-23257653

ABSTRACT

BACKGROUND: Current standard medications for the treatment of allergic inflammation consist primarily of glucocorticoids and anti-histamines, but adverse side effects or insufficient responsiveness by patient subpopulations illustrate the need for safe and novel alternatives. Thus, there is a demand to develop a porcine model that is able to mimic mast cell-mediated type I hypersensitivity. Previously, we found that escin, a pharmacologically active mix of triterpene saponins from horse chestnut extracts, exerts anti-allergic effects in murine models and merits further investigation as an anti-allergic therapeutic. METHODS: We developed a new porcine model of allergic dermatitis based on a clinical prick test protocol. Histamine clearly provoked erythema and swelling at the prick site, whereas the mast cell-degranulating compound 48/80 even more pronounced caused wheal and flare reactions known from the human prick response. This model was used to test the anti-allergic efficacy of orally applied escin. RESULTS: Oral pretreatment of animals with escin strongly inhibited the allergic skin response induced by compound 48/80 in a dose-dependent manner. Additional in vitro data from murine mast cells indicate an engagement of the glucocorticoid receptor pathway upon treatment with escin. CONCLUSIONS: This model provides a valuable and easy-to-set-up tool for preclinical studies of mast cell-inhibiting compounds. The successful implementation of this model supports the development of oral escin applications as a novel anti-allergic therapy.


Subject(s)
Anti-Allergic Agents/pharmacology , Dermatitis, Atopic/drug therapy , Escin/pharmacology , Animals , Disease Models, Animal , Dose-Response Relationship, Immunologic , Female , Histamine/immunology , Male , Skin Tests , Swine , p-Methoxy-N-methylphenethylamine/pharmacology
4.
Cell Host Microbe ; 12(3): 313-23, 2012 Sep 13.
Article in English | MEDLINE | ID: mdl-22980328

ABSTRACT

Nitric oxide (NO) defends against intracellular pathogens, but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with <2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.


Subject(s)
Argininosuccinate Synthase/metabolism , Macrophages/immunology , Macrophages/metabolism , Mycobacterium bovis/immunology , Nitric Oxide/metabolism , Animals , Arginine/metabolism , Argininosuccinate Synthase/genetics , Cells, Cultured , Citrulline/metabolism , Mice
5.
Immunity ; 33(1): 25-34, 2010 Jul 23.
Article in English | MEDLINE | ID: mdl-20637660

ABSTRACT

Transcriptional regulation of the Nos2 gene encoding inducible nitric oxide synthase (iNOS) requires type I interferon (IFN-I) signaling and additional signals emanating from pattern recognition receptors. Here we showed sequential and cooperative contributions of the transcription factors ISGF3 (a complex containing STAT1, STAT2, and IRF9 subunits) and NF-kappaB to the transcriptional induction of the Nos2 gene in macrophages infected with the intracellular bacterial pathogen Listeria monocytogenes. NF-kappaB preceded ISGF3 at the Nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor TFIIH with the associated CDK7 kinase for serine 5 phosphorylation of the RNA polymerase II (pol II) carboxyterminal domain (CTD). Subsequent to TFIIH deposition by NF-kappaB, ISGF3 attracted the pol II enzyme and phosphorylation at CTD S5 occurred. Thus, STATs and NF-kappaB cooperate through pol II promoter recruitment and the phosphorylation of its CTD, respectively, as a prerequisite for productive elongation of iNOS mRNA.


Subject(s)
Listeria monocytogenes , Listeriosis/metabolism , Macrophages/metabolism , Nitric Oxide Synthase Type II/metabolism , Protein Binding , Animals , Cells, Cultured , Cyclin-Dependent Kinases , Gene Expression Regulation, Enzymologic , Interferon Type I/metabolism , Interferon-Stimulated Gene Factor 3/metabolism , Listeriosis/genetics , Listeriosis/pathology , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/genetics , RNA Polymerase II , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Transcription Factor TFIIH , Cyclin-Dependent Kinase-Activating Kinase
6.
PLoS Pathog ; 5(3): e1000355, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19325882

ABSTRACT

Production of type I interferons (IFN-I, mainly IFNalpha and IFNbeta) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized "interferon-producing cell" (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity. In mice infected with L. monocytogenes, systemic IFN-I was amplified via IFN-beta, the IFN-I receptor (IFNAR), and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characteristic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.


Subject(s)
Interferon Type I/biosynthesis , Listeriosis/immunology , Macrophages/immunology , Animals , Antigens, CD/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Flow Cytometry , Interferon Regulatory Factor-7/immunology , Interferon Type I/immunology , Interferon-beta/immunology , Listeria monocytogenes/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Spleen/cytology , Spleen/immunology , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/metabolism
7.
Cell Microbiol ; 10(5): 1116-29, 2008 May.
Article in English | MEDLINE | ID: mdl-18182083

ABSTRACT

Type I IFN (IFN-I) increase the sensitivity of cells and mice to lethal infection with Listeria monocytogenes. Therefore the amount of IFN-I produced during infection might be an important factor determining Listeria virulence. Two commonly used strains of L. monocytogenes, EGD and LO28, were identified as, respectively, low and high inducers of IFN-I synthesis in infected macrophages. Increased IFN-I production resulted from the stronger ability of the LO28 strain to trigger the IRF3 signalling pathway and correlated with an increased sensitization of macrophages to lethal infection. In contrast, stimulation of NFkappaB, MAPK, or inflammasome signalling by the LO28 and EGD strains did not differ significantly. The LO28 strain was more virulent in wild-type (wt) C57/BL6 mice than the EGD strain whereas both strains were similarly virulent in IFN-I receptor-deficient C57/BL6 mice. Together our data suggest that isolates of wt L. monocytogenes differ in their ability to trigger the IRF3 signalling pathway and IFN-I production, and that the amount of IFN-I produced during infection is an important determinant of Listeria virulence.


Subject(s)
Interferon-beta/metabolism , Listeria monocytogenes/pathogenicity , Animals , Bacterial Toxins/metabolism , Heat-Shock Proteins/metabolism , Hemolysin Proteins/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon-beta/genetics , Listeria monocytogenes/metabolism , Listeriosis/microbiology , Macrophages/microbiology , Mice , Mice, Inbred C57BL , RNA, Messenger/metabolism , Receptor, Interferon alpha-beta/genetics , Signal Transduction , Species Specificity , Virulence
8.
J Immunol ; 173(12): 7416-25, 2004 Dec 15.
Article in English | MEDLINE | ID: mdl-15585867

ABSTRACT

Like viruses, intracellular bacteria stimulate their host cells to produce type I IFNs (IFN-alpha and IFN-beta). In our study, we investigated the signals and molecules relevant for the synthesis of and response to IFN by mouse macrophages infected with Listeria monocytogenes. We report that IFN-beta is the critical immediate-early IFN made during infection, because the synthesis of all other type I IFN, expression of a subset of infection-induced genes, and the biological response to type I IFN was lost upon IFN-beta deficiency. The induction of IFN-beta mRNA and the IFN-beta-dependent sensitization of macrophages to bacteria-induced death, in turn, was absolutely dependent upon the presence of the transcription factor IFN regulatory factor 3 (IRF3). IFN-beta synthesis and signal transduction occurred in macrophages deficient for TLR or their adaptors MyD88, TRIF, or TRAM. Expression of Nod2, a candidate receptor for intracellular bacteria, increased during infection, but the protein was not required for Listeria-induced signal transduction to the Ifn-beta gene. Based on our data, we propose that IRF3 is a convergence point for signals derived from structurally unrelated intracellular pathogens, and that L. monocytogenes stimulates a novel TLR- and Nod2-independent pathway to target IRF3 and the type I IFN genes.


Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation/immunology , Interferon-beta/biosynthesis , Intracellular Fluid/immunology , Intracellular Fluid/microbiology , Intracellular Signaling Peptides and Proteins/physiology , Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Transcription Factors/physiology , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/deficiency , Animals , Antigens, Differentiation , Cells, Cultured , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Humans , Interferon Regulatory Factor-3 , Interferon-alpha/biosynthesis , Interferon-alpha/physiology , Interferon-beta/deficiency , Interferon-beta/genetics , Interferon-beta/physiology , Intracellular Fluid/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Listeria monocytogenes/immunology , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Membrane Proteins/deficiency , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88 , Nod2 Signaling Adaptor Protein , Protein Isoforms/biosynthesis , Protein Isoforms/physiology , Receptors, Cell Surface/deficiency , Receptors, Cell Surface/genetics , Receptors, Immunologic/deficiency , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptors , Transcription Factors/deficiency , Transcription Factors/genetics
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