Caspase deficiency alters the murine gut microbiome.

Caspases are aspartate-specific cysteine proteases that have an essential role in apoptosis and inflammation, and contribute to the maintenance of homeostasis in the intestine. These facts, together with the knowledge that caspases are implicated in host-microbe crosstalk, prompted us to investigate the effect of caspase (Casp)1, -3 and -7 deficiency on the composition of the murine gut microbiota. We observed significant changes in the abundance of the Firmicutes and Bacteroidetes phyla, in particular the Lachnospiraceae, Porphyromonodaceae and Prevotellacea families, when comparing Casp-1, -7 and -3 knockout mice with wild-type mice. Our data point toward an intricate relationship between these caspases and the composition of the murine gut microflora.

A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor alpha gene expression.

The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor alpha (TNF-alpha) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF-alpha gene induction by TCR activation is inhibited, whereas virus induction of the TNF-alpha gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-alpha gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor alpha gene promoter.

The human tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-alpha enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-alpha gene transcription and selectively associates with the TNF-alpha promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-alpha transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.

Engagement of tumor necrosis factor (TNF) receptor 1 leads to ATF-2- and p38 mitogen-activated protein kinase-dependent TNF-alpha gene expression.

Engagement of the tumor necrosis factor-alpha (TNF-alpha) receptors by the TNF-alpha ligand results in the rapid induction of TNF-alpha gene expression. The study presented here shows that autoregulation of TNF-alpha gene transcription by selective signaling through tumor necrosis factor receptor 1 (TNFR1) requires p38 mitogen-activated protein (MAP) kinase activity and the binding of the transcription factors ATF-2 and Jun to the TNF-alpha cAMP-response element (CRE) promoter element. Consistent with these findings, TNFR1 engagement results in increased p38 MAP kinase activity and p38-dependent phosphorylation of ATF-2. Furthermore, overexpression of MADD (MAP kinase-activating death domain protein), an adapter protein that binds to the death domain of TNFR1 and activates MAP kinase cascades, results in CRE-dependent induction of TNF-alpha gene expression. Thus, the TNF-alpha CRE site is the target of TNFR1 stimulation and mediates the autoregulation of TNF-alpha gene transcription.

Signaling through the lymphotoxin-beta receptor stimulates HIV-1 replication alone and in cooperation with soluble or membrane-bound TNF-alpha.

The level of ongoing HIV-1 replication within an individual is critical to HIV-1 pathogenesis. Among host immune factors, the cytokine TNF-alpha has previously been shown to increase HIV-1 replication in various monocyte and T cell model systems. Here, we demonstrate that signaling through the TNF receptor family member, the lymphotoxin-beta (LT-beta) receptor (LT-betaR), also regulates HIV-1 replication. Furthermore, HIV-1 replication is cooperatively stimulated when the distinct LT-betaR and TNF receptor systems are simultaneously engaged by their specific ligands. Moreover, in a physiological coculture cellular assay system, we show that membrane-bound TNF-alpha and LT-alpha1beta2 act virtually identically to their soluble forms in the regulation of HIV-1 replication. Thus, cosignaling via the LT-beta and TNF-alpha receptors is probably involved in the modulation of HIV-1 replication and the subsequent determination of HIV-1 viral burden in monocytes. Intriguingly, surface expression of LT-alpha1beta2 is up-regulated on a T cell line acutely infected with HIV-1, suggesting a positive feedback loop between HIV-1 infection, LT-alpha1beta2 expression, and HIV-1 replication. Given the critical role that LT-alpha1beta2 plays in lymphoid architecture, we speculate that LT-alpha1beta2 may be involved in HIV-associated abnormalities of the lymphoid organs.

Polymorphism within the tumor necrosis factor alpha (TNF) promoter region in patients with ankylosing spondylitis.

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the location of the TNF gene in the vicinity of the HLA-B locus, and the prominent role in inflammation of its product, we investigated the association between AS and two G to A transition polymorphisms located at position -238 and -376 in the promoter region of the TNF gene. The distribution of the TNF alleles was determined in 86 HLA-B27+ AS patients and 163 healthy controls. From the 86 AS patients, 33 suffered from acute anterior uveitis (AAU). No significant difference for the TNF-376 polymorphism in AS and healthy controls was observed. The frequency of the TNF-238A allele in HLA-B27+ AS patients was significantly decreased compared to random controls (p = 0.021). However, the frequency of the TNF-238A allele in HLA-B27+ AS patients was not significantly different from that observed in HLA-B27+ healthy individuals (p = 0.6). Assessment of association showed that the TNF-238G allele is in linkage disequilibrium with the HLA-B27 allele (delta = 0.053; P = 0.008). Therefore, we conclude that the association between TNF-238G and AS is secondary to the HLA-B27 gene and that TNF-238 and-TNF-376 alleles are not likely to be involved in the susceptibility to AS.

Functional analysis of a human tumor necrosis factor alpha (TNF-alpha) promoter polymorphism related to joint damage in rheumatoid arthritis.

BACKGROUND: Functional heterogeneity in the tumor necrosis factor alpha (TNF-alpha) gene may be responsible for the TNF-alpha response in infectious and autoimmune diseases. Recently, the TNF-238 promoter polymorphism was observed as being associated with a more destructive disease in rheumatoid arthritis (RA). To determine the relation between TNF-238 and disease progression, the extent of joint destruction in a cohort of 101 RA patients followed for 12 years was analyzed. Furthermore, we have attempted to link this polymorphism to TNF-alpha gene transcription in monocytes and lymphocytes in vitro. PATIENTS, MATERIALS, AND METHODS: The extent of joint destruction determined on X-rays of hands and feet assessed after 0, 3, 6, and 12 years was compared with TNF-238 genotypes. Functional consequences of TNF-alpha gene polymorphisms using reporter gene constructs were analyzed in cells of the monocyte and lymphocyte lineage by means of transient transfection systems. RESULTS: The rate of joint damage in -238GA patients was lower than that in the -238GG patients, independent of HLA-DR4. Damage after 12 years was 76 +/- 30 for the -238GA versus 126 +/- 13 for the -238GG patients as determined by the van der Heijde's modification of Sharp's method. Furthermore, TNF-238A was found to be in linkage disequilibrium with an additional polymorphism at position -376. Functional assays revealed no significant differences in the level of inducible reporter gene expression between the TNF-238/-376 promoter constructs in the cell types tested. CONCLUSION: In a prospective study, we show that the TNF-238GG genotype contributes to progression of joint destruction in RA, independent of the presence of HLA-DR4. However, in vitro transfection assays indicate that TNF-238A by itself or in combination with TNF-376A is not likely to be of direct functional relevance for transcriptional activation. Therefore, these polymorphisms may serve as markers for additional polymorphisms in the TNF/LT locus or neighboring genes that may influence disease severity.

Tumour necrosis factor alpha gene polymorphisms in rheumatoid arthritis: association with susceptibility to, or severity of, disease?

Genetic factors associated with rheumatoid arthritis (RA) might involve variant tumour necrosis factor (TNF)-alpha genes. Therefore, polymorphisms at positions -308, -238, -376, -163 and +70 relative to the transcription initiation site were studied with respect to the susceptibility to, or severity of, RA. TNF-alpha genotypes of 283 RA patients and 116 healthy individuals were determined. Clinical data were obtained from patient files and questionnaires. The distribution of TNF-alpha alleles was similar in RA patients and healthy controls. With respect to disease severity, the TNF-alpha -238GA genotype was found to be associated with the absence of erosions [odds ratio (OR) 4.1, confidence interval 1.0-17]. In addition, this genotype was associated with a lower number of hand joints affected by erosions within the first 3 yr of disease onset compared to -238GG. The association between the -238 polymorphism and radiographic progression was independent of the presence of HLA-DR4. In line with this observation, the OR for the presence of erosions in patients with both risk factors (DR4 and -238GG) compared to patients who lack these factors was 11.1 (1.8-6.8). No associations between the TNF-alpha -308, +70 and -376 alleles and susceptibility to, or severity of, RA could be demonstrated. Our data indicate that the TNF-alpha -238GA genotype is associated with decreased radiologically detectable progression of RA.

Tumor necrosis factor in Plasmodium falciparum malaria: high plasma level is associated with fever, but high production capacity is associated with rapid fever clearance.

In the present study, we investigated 91 patients with Plasmodium falciparum malaria of different severity in a highly endemic area. Patients were examined at least twice daily until clearance of parasites and fever. Plasma cytokine concentrations without and after ex vivo PHA stimulation of whole blood were determined. On admission we found elevated plasma concentrations of TNF, IFN-gamma, and IL-10 compared to levels during and after chemotherapy. Plasma TNF levels on admission were significantly different between patients with severe and mild malaria (differentiated in schoolchildren and adults). The PHA elicited TNF production capacity of peripheral blood leucocytes was suppressed during the acute phase of malaria. High TNF production capacity was associated with faster fever clearance and parasite clearance and, in patients with severe malaria, with higher blood glucose levels. In conclusion we observed circulating TNF concentrations in malaria patients dependent on the severity of disease, which is itself dependent on age, and an association of a high TNF production capacity with parameters for accelerated cure and good prognosis.

TNF-alpha promoter polymorphisms, production and susceptibility to multiple sclerosis in different groups of patients.

TNF-alpha production in whole blood cultures upon stimulation with LPS was determined in 179 individuals from 61 families in order to characterise the magnitude of inherited differences in TNF-alpha production. The three families characterised by highest TNF production showed 7.1 +/- 0.3 ng TNF/ml upon culture with 10 ng LPS and 10.2 +/- 0.2 ng TNF/ml upon culture with 1000 ng LPS. in contrast to the three families characterised by the lowest TNF production that showed a production of 1.6 +/- 0.1 ng TNF upon culture with 10 ng and 2.5 +/- 0.2 ng/ml upon culture with 1000 ng LPS/ml. This difference could not be attributed to the promoter polymorphisms -308 G to A. -238 G to A or -376 G to A, although the -238 GA donors produced 2.1 +/- 0.9 ng TNF upon culture with 10 ng endotoxin compared to 3.2 +/- 2.2 ng TNF for the -238 GG donors. In line with these results the frequency of the -238 GG genotype was increased in hospitalized MS patients in a nursing home (100% 238GG, n = 57) compared to MS patients in an outpatient's clinic (94% 238GG, n = 98) or Dutch controls (90% 238GG, n = 180). These results suggest that the -238 GG genotype is differently distributed in hospitalized MS patients in a nursing home.

Polymorphisms within the human tumor necrosis factor-alpha promoter region in human immunodeficiency virus type 1-seropositive persons.

The question is addressed whether particular tumor necrosis factor-alpha (TNF-alpha) polymorphisms are associated with clinical course and outcome of human immunodeficiency virus type 1 (HIV-1) infection. The distribution of four TNF-alpha guanine (G) to adenosine (A) transition polymorphisms at positions -376, -308, -238, and -163 of the 5' promoter region of the TNF-alpha gene was studied in a nested case-control study among HIV-1-seropositive participants of the Amsterdam Cohort. None of the polymorphisms was significantly associated with long-term asymptomatic survival after HIV-1 infection compared with progression to clinical AIDS. Moreover, specific AIDS-defining illnesses or biologic phenotype of the HIV-1 virus were not associated with TNF-alpha alleles. The results of this study do not point toward a role for known TNF-alpha G to A transition polymorphisms in the clinical course of HIV-1 infection.

Allele-specific quantification of TNFA transcripts in rheumatoid arthritis.

Clinical and laboratory studies have suggested a pivotal role for tumor necrosis factor alpha (TNFA) in the pathogenesis of rheumatoid arthritis (RA). Interindividual variation in the expression of TNFA indicates the existence of functionally distinct TNFA alleles that could play a role in susceptibility to TNFA-associated diseases such as RA. In order to determine whether differential TNFA gene expression is present in RA, we studied the relative contribution of TNFA alleles to the total amount of steady-state mRNA in peripheral blood mononuclear cells of RA patients and healthy individuals. Moreover, allelic TNFA mRNA expression was analyzed in synovial biopsy material of RA patients. For this purpose, we used the recently identified C-insertion polymorphism located in the 5' untranslated region of the first exon. The location of this polymorphism within a part of the gene that is transcribed into mRNA allowed us to discriminate between the contribution of each allele to the total amount of TNFA mRNA in heterozygous individuals. The results of this study do not indicate the existence of variation at the level of mRNA transcribed from each TNFA allele by in vitro and physiological stimulation conditions in RA patients. Therefore, our data do not suggest a role for transcriptionally distinct TNFA alleles in the susceptibility to RA.

The tetracycline derivative minocycline differentially affects cytokine production by monocytes and T lymphocytes.

Minocycline is a tetracycline derivative that has beneficial effects in noninfectious forms of arthritis and dermatitis. To investigate whether this effect may be attributed to interference with cytokine production, we studied the effect of minocycline on cytokine production by T cells and monocytes. Minocycline exerted an inhibitory effect on tumor necrosis factor alpha (TNF-alpha) and gamma interferon production by stimulated T cells, whereas the production of interleukin 6 (IL-6) remained unaffected. The effect of minocycline on TNF-alpha mRNA synthesis by T cells was shown to be stimulus specific. T cells stimulated by a Ca2+-independent mode exhibited a decrease in TNF-alpha mRNA in the presence of minocycline, whereas the TNF-alpha mRNA level remained unaffected by minocycline when cells were stimulated in a Ca2+-dependent manner. In contrast to the effect on T cells, addition of minocycline to lipopolysaccharide-stimulated monocytes led to a dose-dependent increase in TNF-alpha and IL-6 production which was paralleled by an enhancement of TNF-alpha mRNA synthesis. These results indicate that minocycline exerts differential effects on the regulation of cytokine production by T cells and monocytes that are partly reflected at the mRNA level. Given the pleiotropic effects of minocycline, it is suggested that the immunostimulatory effect on monocytes might counteract its beneficial properties in the treatment of several forms of chronic inflammation.

Detection of a C-insertion polymorphism within the human tumor necrosis factor alpha (TNFA) gene.

We have identified a C-insertion polymorphism in the 5'UTR of the first exon of the human tumor necrosis factor alpha (TNFA) gene. TNFA is a cytokine that plays an important role in the inflammatory response.

Relevance of the tumor necrosis factor alpha (TNF alpha) -308 promoter polymorphism in TNF alpha gene regulation.

Tumor necrosis factor alpha (TNF alpha) is a central mediator of the immunological response and the location of the gene within the major histocompatibility complex (MHC) has prompted much speculation about the role of TNF alpha alleles in inflammatory and MHC-associated autoimmune diseases. A G to A transition polymorphism at position -308 of the TNF alpha promoter/enhancer region has been described. The uncommon -308A allele was shown to be strongly associated with human leukocyte antigen (HLA)-DR3, known to be related to a TNF alpha "high producer" phenotype. In support for a clinical relevance, the -308A allele is implicated in susceptibility for cerebral malaria. In this study, we determined the junctional consequences of the TNF -308 polymorphism. Therefore, we analyzed both allelic forms (TNF alpha(-308G) and TNF alpha(-3O8A)) of the TNF alpha enhancer/promoter region (-598/+108) in a transient transfection system, using chloramphenicol acetyltransferase (CAT) as reporter gene. The T cell line Jurkat and the B cell line Raji served as hosts in these experiments. The results showed no differences in the level of inducible reporter gene expression between the TNF(-3O8G)/CAT and the TNF(-308A)/CAT constructs. These data were confirmed by allele specific TNF alpha transcript quantification (ASTQ) analysis, which demonstrated that both TNF alleles contribute equally to the total amount of mRNA in peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/anti-CD3. In analogy, no difference between the level of transcription of the -308A and -308G alleles was observed in lipopolysaccharide (LPS)-stimulated peripheral blood monocytes. This study indicates that the TNF alpha -308 G to A transition is not responsible for differential TNF alpha production induced by standard in vitro stimuli.

Tumor necrosis factor alpha-308 gene variants in relation to major histocompatibility complex alleles and Felty's syndrome.

The location of the human TNF genes within the MHC complex has prompted much speculation about the role of TNF alleles in the etiology of MHC-associated autoimmune diseases. On sequencing the 5' regulatory region of the human TNFA gene a G (TNFA-308G) to A (TNFA-308A) transition polymorphism at position -308 was discovered. We have developed a simple PCR assay to facilitate the screening of the -308 polymorphism at the DNA level. In view of the possible linkage between the TNFA-308A allele and a certain MHC type, TNFA-308 genotypes in HLA-typed healthy individuals (n = 88) were determined. A statistically significant association between the TNFA-308A allele and HLA-DR3, DQB1*0201, DQA1*0501, A1, B8, and the NcoI 5.5-kb RFLP of the TNFB gene was observed. In addition, we determined the frequency of the TNFA-308A allele in patients with FS (n = 13), an HLA-DR4-associated disease. In this study, no association was found of Felty's syndrome with the TNFA-308A allele, indicating that this allele does not appear to be a susceptibility factor for FS.

Expression of granzymes A and B in synovial tissue from patients with rheumatoid arthritis and osteoarthritis.

Granzymes A and B are serine proteinases which are stored in the granules of activated cytotoxic T cells and NK cells. Expression of these granzymes by cytotoxic cells in tissues can be used as an activation marker for these cells. To investigate a possible role of cytotoxic lymphocytes in rheumatoid arthritis (RA) and osteoarthritis (OA), we assessed the expression of granzymes A and B by cytotoxic lymphocytes in synovial biopsies from five RA and five OA patients using mAb specific for these serine proteinases. In three of the five RA patients but also in two of the five OA patients granzyme A- and B-expressing lymphocytes were observed in the synovium. Double-labeling immunohistochemical techniques revealed that up to 75% of the granzyme-positive synovial lymphocytes had the CD16+ or CD56+ natural killer cell phenotype. Less than 5% were CD3+, CD8+ cytotoxic T cells, whereas in some patients the phenotype of up to 50% of these cells could not be identified. The presence of granzymes A and B in the synovium of both RA as well as OA patients was confirmed on the molecular level in a second group of 11 RA and 5 OA patients using the polymerase chain reaction. Thus, expression of granzymes A and B occurs in the synovium in patients with RA as well as those with OA. These proteins are mainly expressed by NK cells that may therefore play a role in the pathogenesis of these diseases.

Polymorphism of tumour necrosis factor-alpha (TNF-alpha) at position -308 in relation to ankylosing spondylitis.

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the localization, in the proximity of the HLA-B locus, and the biological activities of TNF-alpha, we investigated the association between AS and a single base polymorphism located at position -308 of the TNF-alpha gene. An allele-specific polymerase chain reaction was developed to monitor this polymorphism. The frequency of the TNF-alpha alleles was determined in 66 AS patients and 37 healthy controls. The TNF-alpha allele frequency was not significantly different between AS patients and controls.

Detection of intracellular interferon-gamma by light microscopy using an immunoperoxidase technique: correlation with the corresponding mRNA and protein product.

Identifying individual cytokine-producing cells may help to acquire insight into immunological processes. This study was designed to adapt a technique for the detection of individual cytokine-producing cells from an immunofluorescence to an immunoperoxidase staining procedure. The production of interferon-gamma (IFN-gamma) by anti-CD3-activated cloned human T cells was used as a model system. After the conditions for the staining procedure were optimized, the immunoperoxidase technique was slightly more sensitive than the immunofluorescence technique. The intracellular staining for IFN-gamma was preceded or paralleled by IFN-gamma mRNA production and followed by accumulation of IFN-gamma in the supernatant. It is concluded that intracellular IFN-gamma can easily be detected using an immunoperoxidase procedure. This procedure is highly sensitive and allows quantification of the production of multiple cytokines by counting the percentage of positively staining cells.