Regulation of indoleamine 2,3-dioxygenase expression in simian immunodeficiency virus-infected monkey brains

The human immunodeficiency virus type 1-associated cognitive-motor disorder, including the AIDS dementia complex, is characterized by brain functional abnormalities that are associated with injury initiated by viral infection of the brain. Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in tryptophan catabolism in extrahepatic tissues, can lead to neurotoxicity through the generation of quinolinic acid and immunosuppression and can alter brain chemistry via depletion of tryptophan. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we demonstrate that cells of the macrophage lineage are the main source for expression of IDO in the SIV-infected monkey brain. Animals with SIV encephalitis have the highest levels of IDO mRNA, and the level of IDO correlates with gamma interferon (IFN-gamma) and viral load levels. In vitro studies on mouse microglia reveal that IFN-gamma is the primary inducer of IDO expression. These findings demonstrate the link between IDO expression, IFN-gamma levels, and brain pathology signs observed in neuro-AIDS.     

Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection

Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.     

Normal T-cell turnover in sooty mangabeys harboring active simian immunodeficiency virus infection

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) remain healthy though they harbor viral loads comparable to those in rhesus macaques that progress to AIDS. To assess the immunologic basis of disease resistance in mangabeys, we compared the effect of SIV infection on T-cell regeneration in both monkey species. Measurement of the proliferation marker Ki-67 by flow cytometry showed that mangabeys harbored proliferating T cells at a level of 3 to 4% in peripheral blood irrespective of their infection status. In contrast, rhesus macaques demonstrated a naturally high fraction of proliferating T cells (7%) that increased two- to threefold following SIV infection. Ki-67(+) T cells were predominantly CD45RA(-), indicating increased proliferation of memory cells in macaques. Quantitation of an episomal DNA product of T-cell receptor alpha rearrangement (termed alpha1 circle) showed that the concentration of recent thymic emigrants in blood decreased with age over a 2-log unit range in both monkey species, consistent with age-related thymic involution. SIV infection caused a limited decrease of alpha1 circle numbers in mangabeys as well as in macaques. Dilution of alpha1 circles by T-cell proliferation likely contributed to this decrease, since alpha1 circle numbers and Ki-67(+) fractions correlated negatively. These findings are compatible with immune exhaustion mediated by abnormal T-cell proliferation, rather than with early thymic failure, in SIV-infected macaques. Normal T-cell turnover in SIV-infected mangabeys provides an explanation for the long-term maintenance of a functional immune system in these hosts.     

Dynamics of T-cell responses and memory T cells during primary simian immunodeficiency virus infection in cynomolgus macaques

Cellular immune responses make an important contribution to both the control of human immunodeficiency virus (HIV) replication and disease progression. We used a pathogenic model of SIVmac251 infection of cynomolgus macaques to longitudinally evaluate cellular immune responses in association with various rates of disease progression. We found an inverse relationship between plasma viral load and the simian immunodeficiency virus (SIV)-specific T cells responses in peripheral blood and lymph nodes. SIV-specific T-cell responses in peripheral blood were transient during primary infection, with the highest responses detected around 3 months after infection. There was also a transient increase of central memory CD8⁺ T cells in peripheral blood during primary infection, and effector memory T-cell counts in peripheral lymph nodes were increased. This study emphasizes the importance of the early virus-specific immune responses in the outcome of HIV/SIV disease and provides details about the changes of virus-specific immune responses over time.     

Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism

Interferons have been shown to be potential anti-cancer agents and to inhibit tumor cell growth in culture. The in vivo mechanism of the anti-proliferative effect may be direct or indirect through the immune system; however, in vitro a primary mechanism of cytotoxicity is through the depletion of tryptophan. In particular, interferon-gamma (IFN-gamma) induces an enzyme of tryptophan catabolism, indoleamine 2,3-dioxygenase (IDO), which is responsible for conversion of tryptophan and other indole derivatives to kynurenine. The inhibitory effect of interferon on many intracellular parasites such as Toxoplasma gondii and Chlamydia trachomatis is by the same mechanism. Elevated kynurenine levels have been found in humans in a number of diseases and after interferon treatment, and the enzyme is induced in rodents after administration of interferon inducers, or influenza virus. IDO induction also occurs in vivo during rejection of allogeneic tumors, indicating a possible role for this enzyme in the tumor rejection process. The gene for IDO has been cloned and shown to be differentially regulated by IFN-alpha and IFN-gamma. IDO induction has been correlated with induction of GTP-cyclohydrolase, the key enzyme in pteridine biosynthesis. A direct role for IDO in pteridine synthesis has not been shown, and this parallel induction may reflect coordinate regulation of genes induced by IFN-gamma. A possible role for IDO in O2-radical scavenging and in inflammation is discussed.     

The disruption of macaque CD4+ T-cell repertoires during the early simian immunodeficiency virus infection

T-cell receptor (TCR) complementarily determining region 3 (CDR3) spetratyping analysis was employed to assess the ability of an AIDS virus to disrupt CD4 + T-cell repertoires during the primary infection. Rhesus and pig-tailed macaques infected with simian immunodeficiency virus (SIV)mac 251 and SIVsmmFGb, respectively, were evaluated. Following SIV infection, the macaques exhibited an apparent decline of CD4 + peripheral blood lymphocyte (PBL) counts, which was associated with a change in CDR3 profiles from multiple-length distribution to one- or two-length dominance in the selected TCR Vbeta-expressing CD4 + PBL subpopulations. Molecular analysis of the perturbed cell subpopulations suggested that the CD4 + T cells bearing the dominant CDR3 length were clonally expanded. These results indicate that SIV infection can induce a disruption of macaque CD4 + T-cell repertoires during the primary infection. The finding in this study, therefore, suggests that the virus-induced clonal dominance can contribute to the disruption of CD4 + T-cell repertoires.     

Fatal pancreatitis in simian immunodeficiency virus SIV(mac251)-infected macaques treated with 2',3'-dideoxyinosine and stavudine following cytotoxic-T-lymphocyte-associated antigen 4 and indoleamine 2,3-dioxygenase blockade

Human immunodeficiency virus (HIV) infection is associated with immune activation, CD4⁺-T-cell loss, and a progressive decline of immune functions. Antiretroviral therapy (ART) only partially reverses HIV-associated immune dysfunction, suggesting that approaches that target immune activation and improve virus-specific immune responses may be needed. We performed a preclinical study in rhesus macaques infected with the pathogenic simian immunodeficiency virus SIV_mac251 and treated with ART. We tested whether vaccination administered together with cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) blockade and treatment with the indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-d-tryptophan (d-1mT), decreased immune activation and improved vaccine efficacy. The treatment did not augment vaccine immunogenicity; rather, it dramatically increased ART-related toxicity, causing all treated animals to succumb to acute pancreatitis and hyperglycemic coma. The onset of fulminant diabetes was associated with severe lymphocyte infiltration of the pancreas and complete loss of the islets of Langerhans. Thus, caution should be used when considering approaches aimed at targeting immune activation during ART.     

Suppression of cytotoxic and proliferative xenogeneic T-cell responses by transgenic expression of indoleamine 2,3-dioxygenase

Tryptophan catabolism initiated by the enzyme indoleamine 2,3-dioxygenase (IDO) has been postulated to be a natural regulatory mechanism for T cells. In this study, we generated a pig endothelial cell line expressing full-length human IDO (P-HuIDO) to serve as a simple model of a cellular xenogeneic graft. Splenocytes from mice primed to P-HuIDO cells were found to be as responsive to secondary stimulation as splenocytes from mice primed to parental cells. However, in T-cell proliferation assays using P-HuIDO cells as stimulators, a significant inhibition of both naive and memory xenogeneic proliferative responses was noted. Furthermore, the production of interferon-gamma and cytotoxic T lymphocyte function were also affected. When severe combined immunodeficiency mice were grafted with P-HuIDO cells, then challenged with primed splenocytes from BALB/c mice, cellular infiltration to the graft was delayed. Our findings suggest that transgenic expression of IDO in xenografts contributes to prolonged graft survival.     

Inhibition of increased indoleamine 2,3-dioxygenase activity attenuates Toxoplasma gondii replication in the lung during acute infection

The regulation of local L-tryptophan concentrations by tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase (IDO) induced by various stimuli such as interferon-γ (IFN-γ) is one of the key mechanisms in antimicrobial effect. Recently, IDO is also focused on an immunosuppressive mechanism shared by several different immune cell types. Here, we show that inhibition of increased IDO activity maybe involved in the antiparasitic mechanism during Toxoplasma gondii (T. gondii) infection in vivo. In this study, we investigated the role of IDO by using IDO-gene-deficient (IDO KO) mice and by administering a competitive enzyme inhibitor, 1-methyl-D,L-tryptophan (1MT), to wild-type mice following T. gondii infection. Although depletion of lung l-tryptophan did not occur in IDO KO mice after T. gondii infection, the increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) and the inflammatory cytokines in the lung were drastically reduced in the IDO KO mice following infection. We also found that complete depletion of lung l-tryptophan was observed in wild-type mice after infection, but not in mice treated with 1MT. At the same time, 1MT suppressed the increased mRNA expression of SAG2. Taken together, we observed that the inflammatory damage was significantly decreased by the administration of 1MT in the lung after infection. Inhibition of the IDO activity or the elimination of IDO's substrate may be an effective therapy against microbial diseases.     

CD4+ CCR5+ T-cell dynamics during simian immunodeficiency virus infection of Chinese rhesus macaques

Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) provides a reliable model to study the relationship between lentivirus replication, cellular immune responses, and CD4⁺ T-cell dynamics. Here we investigated, using SIVmac251-infected RMs of a Chinese genetic background (which experience a slower disease progression than Indian RMs), the dynamics of CD4⁺ CCR5⁺ T cells, as this subset of memory/activated CD4⁺ T cells is both a preferential target of virus replication and a marker of immune activation. As expected, we observed that the number of circulating CD4⁺ CCR5⁺ T cells decreases transiently at the time of peak viremia. However, at 60 days postinfection, i.e., when set-point viremia is established, the level of CD4⁺ CCR5⁺ T cells was increased compared to the baseline level. Interestingly, this increase correlated with faster disease progression, higher plasma viremia, and early loss of CD4⁺ T-cell function, as measured by CD4⁺ T-cell count, the fraction of memory CD4⁺ T cells, and the recall response to purified protein derivative. Taken together, these data show a key difference between the dynamics of the CD4⁺ CCR5⁺ T-cell pool (and its relationship with disease progression) in Chinese RMs and those described in previous reports for Indian SIVmac251-infected RMs. As the SIV-associated changes in the CD4⁺ CCR5⁺ T-cell pool reflect the opposing forces of SIV replication (which reduces this cellular pool) and immune activation (which increases it), our data suggest that in SIV-infected Chinese RMs the impact of immune activation is more prominent than that of virus replication in determining the size of the pool of CD4⁺ CCR5⁺ T cells in the periphery. As progression of HIV infection in humans also is associated with a relative expansion of the level of CD4⁺ CCR5⁺ T cells, we propose that SIV infection of Chinese RMs is a very valuable and important animal model for understanding the pathogenesis of human immunodeficiency virus infection.     

Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue

Abstract

An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood–brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.     

Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue

An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood-brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.     

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.     

Caspase-dependent and -independent T-cell death pathways in pathogenic simian immunodeficiency virus infection: relationship to disease progression

Studies of human immunodeficiency virus (HIV) and nonhuman primate models of pathogenic and nonpathogenic simian immunodeficiency virus (SIV) infections have suggested that enhanced ex vivo CD4 T-cell death is a feature of pathogenic infection in vivo. However, the relative contributions of the extrinsic and intrinsic pathways to programmed T-cell death in SIV infection have not been studied. We report here that the spontaneous death rate of CD4+ T cells from pathogenic SIVmac251-infected rhesus macaques ex vivo is correlated with CD4 T-cell depletion and plasma viral load in vivo. CD4+ T cells from SIVmac251-infected macaques showed upregulation of the death ligand (CD95L) and of the proapoptotic proteins Bim and Bak, but not of Bax. Both CD4+ and CD8+ T cells from SIVmac251-infected macaques underwent caspase-dependent death following CD95 ligation. The spontaneous death of CD4+ and CD8+ T cells was not prevented by a decoy CD95 receptor or by a broad-spectrum caspase inhibitor (zVAD-fmk), suggesting that this form of cell death is independent of CD95/CD95L interaction and caspase activation. IL-2 and IL-15 prevented the spontaneous death of CD4+ and CD8+ T cells, whereas IL-10 prevented only CD8 T-cell death and IL-7 had no effect on T-cell death. Our results indicate that caspase-dependent and caspase-independent pathways are involved in the death of T cells in pathogenic SIVmac251-infected primates.     

Differential CD4+ T-lymphocyte apoptosis and bystander T-cell activation in rhesus macaques and sooty mangabeys during acute simian immunodeficiency virus infection

In contrast to pathogenic lentiviral infections, chronic simian immunodeficiency virus (SIV) infection in its natural host is characterized by a lack of increased immune activation and apoptosis. To determine whether these differences are species specific and predicted by the early host response to SIV in primary infection, we longitudinally examined T-lymphocyte apoptosis, immune activation, and the SIV-specific cellular immune response in experimentally infected rhesus macaques (RM) and sooty mangabeys (SM) with controlled or uncontrolled SIV infection. SIVsmE041, a primary SIVsm isolate, reproduced set-point viremia levels of natural SIV infection in SM but was controlled in RM, while SIVmac239 replicated to high levels in RM. Following SIV infection, increased CD8⁺ T-lymphocyte apoptosis, temporally coinciding with onset of SIV-specific cellular immunity, and elevated plasma Th1 cytokine and gamma interferon-induced chemokine levels were common to both SM and RM. Different from SM, SIV-infected RM showed a significantly higher frequency of peripheral blood activated CD8⁺ T lymphocytes despite comparable magnitude of the SIV-specific gamma interferon enzyme-linked immunospot response. Furthermore, an increase in CD4⁺ and CD4⁻CD8⁻ T-lymphocyte apoptosis and plasma tumor necrosis factor-related apoptosis-inducing ligand were observed only in RM and occurred in both controlled SIVsmE041 and uncontrolled SIVmac239 infection. These data suggest that the “excess” activated T lymphocytes in RM soon after SIV infection are predominantly of non-virus-specific bystander origin. Thus, species-specific differences in the early innate immune response appear to be an important factor contributing to differential immune activation in natural and nonnatural hosts of SIV infection.     

Transient antiretroviral treatment during acute simian immunodeficiency virus infection facilitates long-term control of the virus

Experimental evidence and mathematical models indicate that CD4+ T-cell help is required to generate memory cytotoxicT-lymphocyte precursors (CTLp) that are capable of persisting without ongoing antigenic stimulation, and that such responses are necessary to clear an infection or to control it in the long term. Here we analyse mathematical models of simian immunodeficiency virus (SIV) replication in macaques, assuming that SIV impairs specific CD4+ T-cell responses. According to the models, fast viral replication during the initial stages of primary infection can result in failure to generate sufficient long-lived memory CTLp required to control the infection in the long term. Modelling of drug therapy during the acute phase of the infection indicates that transient treatment can minimize the amount of virus-induced immune impairment, allowing a more effective initial immune sensitization. The result is the development of high levels of memory CTLp that are capable of controlling SIV replication in the long term, in the absence of continuous treament. In the model, the success of treatment depends crucially on the timing and duration of antiretroviral therapy. Data on SIV-infected macaques receiving transient drug therapy during acute infection support these theoretical predictions. The data and modelling suggest that among subjects controlling SIV replication most efficiently after treatment, there is a positive correlation between cellular immune responses and virus load in the post-acute stage of infection. Among subjects showing less-efficient virus control, the correlation is negative. We discuss our findings in relation to previously published data on HIV infection.     

Conditional CD8+ T cell escape during acute simian immunodeficiency virus infection

CD8⁺ T cell responses rapidly select viral variants during acute human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. We used pyrosequencing to examine variation within three SIV-derived epitopes (Gag_386-394GW9, Nef_103-111RM9, and Rev_59-68SP10) targeted by immunodominant CD8⁺ T cell responses in acutely infected Mauritian cynomolgus macaques. In animals recognizing all three epitopes, variation within Rev_59-68SP10 was associated with delayed accumulation of variants in Gag_386-394GW9 but had no effect on variation within Nef_103-111RM9. This demonstrates that the entire T cell repertoire, rather than a single T cell population, influences the timing of immune escape, thereby providing the first example of conditional CD8⁺ T cell escape in HIV/SIV infection.     

Memory CD4+ T-lymphocyte loss and dysfunction during primary simian immunodeficiency virus infection

It has long been appreciated that CD4+ T lymphocytes are dysfunctional in human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV)-infected individuals, and it has recently been shown that HIV/SIV infections are associated with a dramatic early destruction of memory CD4+ T lymphocytes. However, the relative contributions of CD4+ T-lymphocyte dysfunction and loss to immune dysregulation during primary HIV/SIV infection have not been fully elucidated. In the current study, we evaluated CD4+ T lymphocytes and their functional repertoire during primary SIVmac251 infection in rhesus monkeys. We show that the extent of loss of memory CD4+ T lymphocytes and staphylococcal enterotoxin B-stimulated cytokine production by total CD4+ T lymphocytes during primary SIVmac251 infection is tightly linked in a cohort of six rhesus monkeys to set point plasma viral RNA levels, with greater loss and dysfunction being associated with higher steady-state viral replication. Moreover, in exploring the mechanism underlying this phenomenon, we demonstrate that the loss of functional CD4+ T lymphocytes during primary SIVmac251 infection is associated with both a selective depletion of memory CD4+ T cells and a loss of the functional capacity of the memory CD4+ T lymphocytes that escape viral destruction.