Milton W. Taylor

Indoleamine 2,3-Dioxygenase Production by Human Dendritic Cells Results in the Inhibition of T Cell Proliferation

Dendritic cells (DCs) play a key role in the activation and regulation of B and T lymphocytes. Production of indoleamine 2,3-dioxygenase (IDO) by macrophages has recently been described to result in inhibition of T cell proliferation through tryptophan degradation. Since DCs can be derived from monocytes, we sought to determine whether DCs could produce IDO which could potentially regulate T cell proliferation. Northern blot analysis of RNA from cultured monocyte-derived human DC revealed that IDO mRNA was induced upon activation with CD40 ligand and IFN-γ. IDO produced from activated DCs was functionally active and capable of metabolizing tryptophan to kynurenine. Activated T cells were also capable of inducing IDO production by DCs, which was inhibited by a neutralizing Ab against IFN-γ. DC production of IDO resulted in inhibition of T cell proliferation, which could be prevented using the IDO inhibitor 1-methyl-dl-tryptophan. These results suggest that activation of DCs induces the production of functional IDO, which causes depletion of tryptophan and subsequent inhibition of T cell proliferation. This may represent a potential mechanism for DCs to regulate the immune response.

Changes in Gene Expression during Pegylated Interferon and Ribavirin Therapy of Chronic Hepatitis C Virus Distinguish Responders from Nonresponders to Antiviral Therapy

ABSTRACT Treating chronic hepatitis C virus (HCV) infection using pegylated alpha interferon and ribavirin leads to sustained clearance of virus and clinical improvement in approximately 50% of patients. Response rates are lower among patients with genotype 1 than with genotypes 2 and 3 and among African-American (AA) patients compared to Caucasian (CA) patients. Using DNA microarrays, gene expression was assessed for a group of 33 African-American and 36 Caucasian American patients with chronic HCV genotype 1 infection during the first 28 days of treatment. Results were examined with respect to treatment responses and to race. Patients showed a response to treatment at the gene expression level in RNA isolated from peripheral blood mononuclear cells irrespective of degree of decrease in HCV RNA levels. However, gene expression responses were relatively blunted in patients with poor viral response (<1.5 log10-IU/ml decrease at 28 days) compared to those in patients with a marked (>3.5 log10-IU/ml decrease) or intermediate (1.5 to 3.5 log10-IU/ml decrease) response. The number of genes that were up- or down-regulated by pegylated interferon and ribavirin treatment was fewer in patients with a poor response than in those with an intermediate or marked viral response. However AA patients had a stronger interferon response than CA patients in general. The induced levels of known interferon-stimulated genes such as the 2′5′-oligoadenylate synthetase, MX1, IRF-7, and toll-like receptor TLR-7 genes was lower in poor-response patients than in marked- or intermediate-response patients. Thus, the relative lack of viral response to interferon therapy of hepatitis C virus infection is associated with blunted interferon cell signaling. No specific regulatory gene could be identified as responsible for this global blunting or the racial differences.

Pretreatment Sequence Diversity Differences in the Full-Length Hepatitis C Virus Open Reading Frame Correlate with Early Response to Therapy

ABSTRACT Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.

Importance of the Two Interferon-stimulated Response Element (ISRE) Sequences in the Regulation of the Human Indoleamine 2,3-Dioxygenase Gene

Indoleamine 2,3-dioxygenase (INDO) is the rate-limiting enzyme in the catabolism of the essential amino acid L-tryptophan. It is induced strongly in many cell lines following interferon-γ treatment. We report the cloning and characterization of the full-length human INDO promoter. This promoter is 1,245 base pairs long and includes two interferon-stimulated response elements (ISRE) separated by an approximately 1-kilobase sequence. The presence of these two ISREs is critical for maximum INDO promoter activity (50-fold induction). When the ISREs are present in two separate fragments cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter vector, the INDO promoter activity drops significantly (7-fold induction). 5′ end deletions of the wild type promoter sequence indicate that removal of the ISRE (ISRE1) at position −1126 reduces the induction level to approximately 25-fold. This activity does not change appreciably when the promoter is deleted down to position −241. Furthermore, site-directed mutagenesis of ISRE1 also decreases the promoter activity in a similar way. When ISRE1 is kept intact, deletion of the second ISRE (ISRE2) at position −111 leads to only 11-fold induction of the promoter. A similar result is obtained when substitution mutations are introduced in ISRE2. Deletion of a 748-base pair sequence between the two ISREs only shows a slight decrease in the INDO promoter activity. These data indicate that the two ISRE sequences are required for the full transcriptional induction of the interferon-γ-inducible human INDO gene. INDO activity is not induced in the hepatic cell line HepG2. An analysis of INDO-CAT activity in this cell line indicated that the lack of INDO activity was at the transcriptional level and could reflect either the presence of a repressor or lack of a transcription factor. This lack of induction could be correlated with a truncated or unstable IRF-1. However, the levels of IRF-2, JAK2, and STAT 91 were similar in both ME180 and HepG2 cells.

Global Effect of PEG-IFN-α and Ribavirin on Gene Expression in PBMC In Vitro

Using oligonucleotide microarrays, we have examined the expression of 22,000 genes in peripheral blood cells treated with pegylated interferon-alpha2b (PEG-IFN-alpha) and ribavirin. Treatment with ribavirin had very little effect on gene expression, whereas treatment with PEG-IFN-alpha had a dramatic effect, modulating the expression of approximately 1000 genes (at p < 0.001). In addition to genes previously reported to be induced by type I or type II IFNs, many novel genes were found to be upregulated, including transcription factors, such as ATF3, ATF4, properdin, a key regulator of the complement pathway, a homeobox gene (HESX1), and an RNA editing enzyme (apobec3). Chemokines CXCL10 and CXCL11 were upregulated, whereas CXCL5 was downregulated. Cytokines interleukin-15 (IL-15) and IL-18 were also significantly induced, whereas IL-1alpha and IL-1beta were downregulated. Most other interleukins were not affected. The results of the microarrays were confirmed by kinetic real-time PCR. These data indicate that IFN treatment causes upregulation of genes associated with the stress response, apoptosis, and signaling, and an equal number of genes are downregulated, including those associated with protein synthesis, specific cytokines and chemokines and other biosynthetic functions.

Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-β: Selective inhibition of indoleamine 2,3- dioxygenase and tryptophanyl-tRNA synthetase gene expression

Alterations in the rate of cellular tryptophan metabolism are involved in mediating important biological activities associated with cytokines and growth factors. Indoleamine 2,3‐dioxygenase (IDO) and tryptophanyl‐tRNA synthetase are enzymes of tryptophan metabolism whose expression in a variety of cells and tissues is highly inducible by interferon‐γ (IFN‐γ). Transforming growth factor‐β (TGF‐β) antagonizes many cellular responses to IFN‐γ. The interaction of these two cytokines plays an important role in maintaining homeostasis during inflammation and repair. In human skin and synovial fibroblasts in vitro, TGF‐β caused time‐ and dose‐dependent abrogation of IFN‐γ‐stimulated expression of IDO and tryptophanyl‐tRNA synthetase mRNAs. The inhibition was selective and did not appear to be due to down‐regulation of IFN‐γ signaling by TGF‐β. In parallel with its effect on IDO mRNA expression, TGF‐β caused a marked reduction in intracellular IDO protein levels and abrogated IDO activity and tryptophan catabolism in these cells induced by IFN‐γ. IFN‐γ caused a rapid and striking increase in the amount of IDO heterogeneous nuclear pre‐mRNA and induced transcription of the IDO gene, as demonstrated by transient transfection assays. TGF‐β partially reversed this stimulation. IFN regulatory factor (IRF)‐1 and stat1 are cellular intermediates in IFN signaling. Both are implicated in activation of IDO transcription in response to IFN‐γ. The stimulation by IFN‐γ of IRF‐1 protein and mRNA expression was not prevented by treatment of fibroblasts with TGF‐β. Furthermore, gel mobility shift assays indicated that TGF‐β did not inhibit the induction of stat1 and IRF‐1 binding activity to their cognate DNA recognition sites in the IDO gene promoter. In contrast, the stability of IDO mRNA transcripts was reduced in fibroblasts treated with TGF‐β, as shown by determination of mRNA half‐lives following blockade of transcription with 5,6‐dichlorobenzimidazole riboside. The findings indicate that TGF‐β prevents the induction of IDO and tryptophanyl‐tRNA synthetase gene expression in fibroblasts. The repression of IDO expression by TGF‐β is mediated at both transcriptional and posttranscriptional levels. These results implicate TGF‐β in the negative regulation of tryptophan metabolism, provide evidence for the molecular basis of this regulation, and indicate that cellular tryptophan metabolism is under tight immunological control. J. Cell. Physiol. 177:174–186, 1998.

Natural killer inhibitory receptor expression associated with treatment failure and interleukin‐28B genotype in patients with chronic hepatitis C

Natural killer (NK) cells constitute a first line of defense against viral infections; their function is governed by the integration of signals from multiple activating and inhibitory surface receptors. We hypothesized that because NKs become rapidly activated by cytokines, response to anti‐hepatitis C virus (HCV) therapy would be predicted by the phenotype and function of NKs. We used a cohort of 101 patients (55 African, 46 Caucasian‐American) who received pegylated‐interferon (IFN) and ribavirin for 48 weeks. Multiparameter FACS analysis was used to examine relative expression of 14 different inhibitory/activating receptors. Interleukin (IL)‐28B genotyping (rs12979860) was also performed. Pretreatment levels of inhibitory receptors CD158a, CD158b, and CD158e were higher in patients who demonstrated poor viral decline within the first 28 days of therapy. Higher expression levels of inhibitory receptors NKG2A, CD158b, and CD158e were demonstrable in patients who failed to achieve sustained virologic response (SVR). Patients carrying the IL‐28B T allele had higher NKG2A expression on effector NKs. We created a mathematical regression model incorporating race, viral level, and two inhibitory receptors. The area‐under‐the curve was 0.88, which is highly predictive of SVR. Moreover, the model performed complementarily with IL‐28B across the CC, CT, and TT genotypes. Purified NKG2Aneg NKs treated with pegylated‐IFN‐α for 4 hours demonstrated higher levels of IFN‐γ‐inducible protein‐10 (IP‐10) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) compared with their NKG2Apos counterparts. Conclusions: These results provide novel insights into the associations of NK phenotype with IL‐28B genotype and gene expression patterns, as well as the role of NKs in mediating IFN‐induced viral clearance of chronic HCV infection. (HEPATOLOGY 2011;)

Analysis of Transcription Factors Regulating Induction of Indoleamine 2,3-Dioxygenase by IFN-gamma

IFN-gamma treatment of the human carcinoma cell line ME180 causes cell death due to induction of indoleamine 2,3-dioxygenase (IDO) and resulting starvation for tryptophan. A mutant cell line 3B6A derived from ME180 was resistant to IFN-gamma because of loss of IDO activity. Cotransfecting an IDO promoter-chloramphenicol acetyl transferase (CAT) construct with IFN regulatory factor-1 (IRF-1) resulted in induction of CAT activity in both ME180 and 3B6A cells even in the absence of IFN-gamma. This induction was reduced by cotransfection with IRF-2. However, IRF-1 was not able to restore IDO activity, suggesting a possible repressor site outside the IDO promoter region. Stat1alpha (p91) restored both CAT and IDO activities in 3B6A cells following IFN-gamma treatment. 3B6A cells doubly treated with IFN-gamma and IFN-alpha or IFN-beta restored IDO activity, although neither cytokine on its own could induce IDO. Western blot analysis showed that both constitutive expression and induction of Stat1alpha by IFN-gamma were reduced in 3B6A cells, and double treatment of IFN-gamma with IFN-alpha or IFN-beta restored the expression level of Statla. Electrophoretic mobility shift assays indicated that Stat1 binds to the IFN-gamma-activated sequence (GAS) region in the IDO promoter in ME180 cells following IFN-gamma treatment. Our results indicated that the defect in 3B6A cells was reduced expression of Stat1alpha and that IRF-1, NF-kappaB, and PKR were all involved to some extent in the induction of IDO following IFN-gamma treatment.

The effects of interferon on the expression of human papillomavirus oncogenes

The effects of interferon (IFN)-alpha, IFN-beta and IFN-gamma on human papillomavirus (HPV) oncogene expression were studied in various cervical carcinoma cell lines containing integrated copies of either HPV type 16 or HPV type 18. The levels of E6 and E7 transcripts were examined 6 h and 30 h after treatment with IFN. In HeLa cells, all three classes of IFNs effected a decrease in the level of HPV-18 E6 and E7 transcripts. On the other hand, none of the IFNs altered the level of these transcripts in C-4II cells. Only IFN-gamma decreased the level of HPV-16 E6 and E7 transcripts in CaSki and HPK1A cells, while IFN-gamma actually increased the level of these transcripts in SiHa cells. This differential IFN regulation of HPV expression in various cervical cancer cell lines may account for the contradictory clinical results observed after treatment of cervical cancer with IFN.

Correction of a deletion mutant by gene targeting with an adenovirus vector.

The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.