scholarly journals Screening for Toxoplasma gondii-Regulated Transcriptional Responses in Lipopolysaccharide-Activated Macrophages

2006 ◽  
Vol 74 (3) ◽  
pp. 1916-1923 ◽  
Author(s):  
Chiang W. Lee ◽  
Soumaya Bennouna ◽  
Eric Y. Denkers
Keyword(s):  
Toxoplasma Gondii ◽  
Defense Mechanisms ◽  
Transcriptional Response ◽  
Interleukin 12 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Mediated Immunity ◽  
Transcriptional Responses ◽  
Necrosis Factor Alpha ◽  
Reverse Transcription Pcr ◽  
Tnf Α

ABSTRACT Toxoplasma gondii-infected macrophages are blocked in production of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) upon activation with lipopolysaccharide (LPS). Here, we used pathway-focused cDNA arrays to identify additional T. gondii-regulated transcriptional responses. Parasite infection decreased 57 (inclusive of IL-12 and TNF-α) and increased expression of 7 of 77 LPS-activated cytokine and cytokine-related genes. Interestingly, we found that the LPS-induced transcriptional response of the anti-inflammatory cytokine IL-10 was synergistically increased by T. gondii, results that we validated by conventional reverse transcription-PCR and enzyme-linked immunosorbent assay. Importantly, although the parasite exerted disparate effects in LPS-signaling leading to TNF-α versus IL-10 production, both responses required functional Toll-like receptor 4. We suggest that these effects represent parasite defense mechanisms to avoid or delay induction of antimicrobial activity and/or T-cell-mediated immunity during Toxoplasma infection.

scholarly journals Respiratory Tract Infection with Mycoplasma pneumoniae in Interleukin-12 Knockout Mice Results in Improved Bacterial Clearance and Reduced Pulmonary Inflammation

2006 ◽  
Vol 75 (1) ◽  
pp. 236-242 ◽  
Author(s):  
C. M. Salvatore ◽  
M. Fonseca-Aten ◽  
K. Katz-Gaynor ◽  
A. M. Gomez ◽  
A. Mejias ◽  
...  
Keyword(s):  
Respiratory Disease ◽  
Mycoplasma Pneumoniae ◽  
Pulmonary Inflammation ◽  
Airway Hyperreactivity ◽  
Interleukin 12 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 in M. pneumoniae respiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 107 CFU of M. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected with M. pneumoniae were found to have significantly lower BAL M. pneumoniae concentrations compared with M. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-γ than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P = 0.065) and TNF-α (P = 0.078). No differences were found for IL-1β, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimental M. pneumoniae pneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.

scholarly journals Mechanism of Entry Determines the Ability of Toxoplasma gondii To Inhibit Macrophage Proinflammatory Cytokine Production

2002 ◽  
Vol 70 (9) ◽  
pp. 5216-5224 ◽  
Author(s):  
Barbara A. Butcher ◽  
Eric Y. Denkers
Keyword(s):  
Toxoplasma Gondii ◽  
Cytokine Production ◽  
Parasitophorous Vacuole ◽  
Interleukin 12 ◽  
Proinflammatory Cytokine Production ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Infected Cells ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT Macrophages (Mφ) infected with tachyzoites of the opportunistic protozoan Toxoplasma gondii are blocked in production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12) in response to lipopolysaccharide (LPS) triggering, and this is associated with parasite-induced inhibition of NFκB translocation. Here, we demonstrate a requirement for active invasion in the ability of the parasite to mediate suppression. Neither soluble tachyzoite antigen nor secreted products were suppressive, and heat-inactivated, antibody-coated tachyzoites, which efficiently entered the cell through receptor-mediated uptake, failed to inhibit LPS responses. Cytochalasin D, a drug blocking tachyzoite invasion of, but not adherence to, Mφ, severely curtailed Toxoplasma-induced suppression. In addition, parasite-induced nonresponsiveness, as measured by TNF-α production, was reversed by treating infected cells with the toxoplasmastatic drugs pyrimethamine and 6-thioxanthine prior to LPS stimulation. A divergence in IL-12 and TNF-α responses was found during extended incubation of tachyzoites and Mφ in that 24 h of incubation of infected Mφ resulted in IL-12, but not TNF-α, secretion, and production of the latter cytokine remained suppressed when these cells were subjected to LPS triggering. Our results demonstrate that active invasion and survival of the parasite within the parasitophorous vacuole are required to induce and maintain Mφ cytokine-specific nonresponsiveness to LPS. They also show that the effects of Toxoplasma on IL-12 and TNF-α production are nonidentical, with the parasite exerting a longer-lasting suppression of the latter.

scholarly journals Induction of Interleukin-12 and Gamma Interferon Requires Tumor Necrosis Factor Alpha for Protective T1-Cell-Mediated Immunity to Pulmonary Cryptococcus neoformans Infection

2002 ◽  
Vol 70 (6) ◽  
pp. 2959-2964 ◽  
Author(s):  
Amy C. Herring ◽  
John Lee ◽  
Roderick A. McDonald ◽  
Galen B. Toews ◽  
Gary B. Huffnagle
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Interleukin 12 ◽  
Cell Mediated Immunity ◽  
Pulmonary Eosinophilia ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha ◽  
Ifn Γ ◽  
Necrosis Factor

ABSTRACT The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-α) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-α expression was detected in lavage sample cells at days 2, 3, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-α-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-α prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-α monoclonal antibody (day 0) diminished the lung levels of TNF-α, interleukin-12 (IL-12), and gamma interferon (IFN-γ) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-α (day 0) also altered the IFN-γ/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-α-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-α-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-γ in response to the infection. These findings demonstrate that afferent-phase TNF-α production is essential for the induction of IL-12 and IFN-γ and neutralization of early TNF-α results in a T2 shift of the T1/T2 balance of antifungal immunity.

scholarly journals Nramp1 Is Not a Major Determinant in the Control of Brucella melitensis Infection in Mice

2003 ◽  
Vol 71 (2) ◽  
pp. 621-628 ◽  
Author(s):  
Laurence A. Guilloteau ◽  
Jacques Dornand ◽  
Antoine Gross ◽  
Michel Olivier ◽  
Fabienne Cortade ◽  
...  
Keyword(s):  
Brucella Melitensis ◽  
Natural Resistance ◽  
Interleukin 12 ◽  
Necrosis Factor Alpha ◽  
Reverse Transcription Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Oxide Synthase

ABSTRACT Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1s ) and C.CB congenic mice (Nramp1r ) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by Brucella. However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene appears to be of limited importance for the natural resistance of mice to Brucella.

scholarly journals Neutrophil Depletion during Toxoplasma gondii Infection Leads to Impaired Immunity and Lethal Systemic Pathology

2001 ◽  
Vol 69 (8) ◽  
pp. 4898-4905 ◽  
Author(s):  
Susan K. Bliss ◽  
L. Cristina Gavrilescu ◽  
Ana Alcaraz ◽  
Eric Y. Denkers
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Toxoplasma Gondii ◽  
Parasite Burden ◽  
Interleukin 12 ◽  
Necrosis Factor Alpha ◽  
Reverse Transcription Pcr ◽  
Neutrophil Depletion ◽  
Toxoplasma Gondii Infection ◽  
Systemic Pathology

ABSTRACT The immunomodulatory role of neutrophils during infection withToxoplasma gondii was investigated. Monoclonal antibody-mediated depletion revealed that neutrophils are essential for survival during the first few days of infection. Moreover, neutrophil depletion was associated with a weaker type 1 immune response as measured by decreased levels of gamma interferon, interleukin-12 (IL-12) and tumor necrosis factor alpha. IL-10 was also decreased in depleted animals. Additionally, splenic populations of CD4+T cells, CD8+ T cells, and NK1.1+ cells were decreased in depleted mice. Neutrophil-depleted mice exhibited lesions of greater severity in tissues examined and a greater parasite burden as determined by histopathology and reverse transcription-PCR. We conclude that neutrophils are critical near the time of infection because they influence the character of the immune response and control tachyzoite replication.

scholarly journals Abundant Secretion of Bioactive Interleukin-1β by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

2005 ◽  
Vol 73 (1) ◽  
pp. 453-458 ◽  
Author(s):  
P. Kelk ◽  
R. Claesson ◽  
L. Hänström ◽  
U. H. Lerner ◽  
S. Kalfas ◽  
...  
Keyword(s):  
Actinobacillus Actinomycetemcomitans ◽  
Interleukin 1Β ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Human Macrophages ◽  
Specific Antibodies ◽  
Protein Levels ◽  
Caspase 1 ◽  
Reverse Transcription Pcr ◽  
Tnf Α

ABSTRACT Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1β, while the effects on IL-6 and TNF-α production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1β than did LPS (100 ng/ml). The secreted IL-1β was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1β. The presence of specific antibodies to IL-1β or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1β. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1β by human macrophages, which is mediated by activation of caspase 1.

scholarly journals Intranasal Interleukin-12 Therapy Inhibits Mycoplasma pneumoniae Clearance and Sustains Airway Obstruction in Murine Pneumonia

2007 ◽  
Vol 76 (2) ◽  
pp. 732-738 ◽  
Author(s):  
C. M. Salvatore ◽  
M. Fonseca-Aten ◽  
K. Katz-Gaynor ◽  
A. M. Gomez ◽  
R. D. Hardy
Keyword(s):  
Airway Obstruction ◽  
Mycoplasma Pneumoniae ◽  
Statistical Significance ◽  
Airway Hyperreactivity ◽  
Interleukin 12 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Strong Trend ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentrations in respiratory secretions. We evaluated the effects of IL-12 therapy on microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in mice. BALB/c mice were inoculated with M. pneumoniae or SP4 broth. Mice were treated with intranasal IL-12 or placebo daily for 8 days, starting on day 1 after inoculation. Mice were evaluated at baseline and on days 1, 3, 6, and 8 after therapy. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic score (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and granulocyte-macrophage colony-stimulating factor), and plethysmography, both before and after methacholine treatment. M. pneumoniae-infected mice treated with IL-12 (MpIL12 mice) were found to have significantly higher BAL M. pneumoniae concentrations than those of M. pneumoniae-infected mice treated with placebo (MpP mice) (P < 0.001). MpIL12 mice had higher BAL concentrations of IL-12, IFN-γ, TNF-α, and IL-6, with differences in IL-12 and IFN-γ concentrations reaching statistical significance (P < 0.001). Airway obstruction was statistically elevated in MpIL12 mice compared to that in MpP mice (P = 0.048), while airway hyperreactivity was also elevated in MpIL12 mice but did not reach statistical significance (P = 0.081). Lung parenchymal pneumonia subscores were significantly higher in MpIL12 mice (P < 0.001), but no difference was found for overall HPS, even though a strong trend was noticed (P = 0.051). Treatment of experimental M. pneumoniae pneumonia with intranasal IL-12 was associated with more severe pulmonary disease and less rapid microbiologic and histological resolution.

scholarly journals Different Strains of Toxoplasma gondii Induce Different Cytokine Responses in CBA/Ca Mice

2003 ◽  
Vol 71 (7) ◽  
pp. 4171-4174 ◽  
Author(s):  
Fausto G. Araujo ◽  
Teri Slifer
Keyword(s):  
Toxoplasma Gondii ◽  
Toxoplasmic Encephalitis ◽  
Interleukin 12 ◽  
Type Ii ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha ◽  
Ifn Γ ◽  
Different Strains ◽  
Necrosis Factor

ABSTRACT To investigate the role that cytokines may have in the development of toxoplasmic encephalitis (TE), the levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12 ]p40[), IL-10, IL-6, IL-4, and IL-2 in serum were examined in CBA/Ca mice infected with a type II strain (ME49 or FORT) of Toxoplasma gondii. These strains caused severe (ME49) or mild (FORT) TE in CBA/Ca mice. From weeks 1 to 8 of infection, the levels of IL-6, IL-10, IL-12, IFN-γ, and TNF-α in serum were significantly higher in the ME49-infected mice than in the FORT-infected mice, suggesting a role for these cytokines in the severity of TE in CBA/Ca mice. Since the ME49 and FORT strains are of the same type, our results suggest a role for the parasite in the development of severe TE through the increased production of proinflammatory cytokines and indicate that not all type II strains cause TE.

scholarly journals Guinea Pig Neutrophils Infected with Mycobacterium tuberculosis Produce Cytokines Which Activate Alveolar Macrophages in Noncontact Cultures

2007 ◽  
Vol 75 (4) ◽  
pp. 1870-1877 ◽  
Author(s):  
Kirti V. Sawant ◽  
David N. McMurray
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pig ◽  
Alveolar Macrophages ◽  
Early Period ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Independent Manner ◽  
Necrosis Factor Alpha ◽  
Reverse Transcription Pcr ◽  
Tnf Α

ABSTRACT The early influx of neutrophils to the site of infection may be an important step in host resistance against Mycobacterium tuberculosis. In this study, we investigated the effect of M. tuberculosis infection on the ability of guinea pig neutrophils to produce interleukin-8 (IL-8; CXCL8) and tumor necrosis factor alpha (TNF-α) and to activate alveolar macrophages. Neutrophils and alveolar macrophages were isolated from naïve guinea pigs, cultured together or alone, and infected with virulent M. tuberculosis for 3, 12, and 24 h. IL-8 protein production in cocultures, as measured by using an enzyme-linked immunosorbent assay, was found to be additive at 24 h and significantly greater in M. tuberculosis-infected cocultures than in uninfected cocultures and in cultures of the infected neutrophils or macrophages alone. The IL-8 mRNA levels, determined by real-time reverse transcription-PCR, were elevated at 24 h in infected cocultures and infected cells cultured alone. In order to elucidate the contributions of neutrophils and their soluble mediators to the activation of alveolar macrophages, neutrophils and alveolar macrophages were cultured in a contact-independent manner by using a Transwell insert system. Neutrophils were infected with virulent M. tuberculosis in the upper wells, and alveolar macrophages were cultured in the lower wells. The release of hydrogen peroxide from alveolar macrophages exposed to soluble products from infected neutrophils was significantly increased compared to that from unexposed alveolar macrophages. Significant up-regulation of IL-1β and TNF-α mRNA levels in alveolar macrophages was observed at 24 and 30 h, respectively, compared to those in cells not exposed to soluble neutrophil products. Treatment with anti-guinea pig TNF-α polyclonal antibody completely abolished the response of alveolar macrophages to neutrophil products. This finding suggests that TNF-α produced by infected neutrophils may be involved in the activation of alveolar macrophages and hence may contribute to the containment of M. tuberculosis infection during the early period of infection.

Morin Promotes the Production of Th2 Cytokine by Modulating Bone Marrow-Derived Dendritic Cells

2006 ◽  
Vol 34 (04) ◽  
pp. 667-684 ◽  
Author(s):  
Chia-Yang Li ◽  
Jau-Ling Suen ◽  
Bor-Luen Chiang ◽  
Pei-Dawn Lee Chao ◽  
Shih-Hua Fang
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
Interleukin 12 ◽  
Th2 Response ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Th Differentiation ◽  
Th1 And Th2 Responses

Our previous studies had reported that morin decreased the interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS)-activated macrophages, suggesting that morin may promote helper T type 2 (Th2) response in vivo. Dendritic cells (DCs) are the most potent antigen presenting cells and known to play a major role in the differentiation of helper T type 1 (Th1) and Th2 responses. This study aimed to reveal whether morin is able to control the Th differentiation through modulating the maturation and functions of DCs. Bone marrow-derived dendritic cells (BM-DCs) were incubated with various concentrations of morin and their characteristics were studied. The results indicated that morin significantly affects the phenotype and cytokine expression of BM-DCs. Morin reduced the production of IL-12 and TNF-α in BM-DCs, in response to LPS stimulation. In addition, the proliferative response of stimulated alloreactive T cells was significantly decreased by morin in BM-DCs. Furthermore, allogeneic T cells secreted higher IL-4 and lower IFN-γ in response to morin in BM-DCs. In conclusion, these results suggested that morin favors Th2 cell differentiation through modulating the maturation and function of BM-DCs.

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