Induction of indoleamine 2,3-dioxygenase in primary human macrophages by HIV-1

Abstract

Increased kynurenine pathway metabolism has been implicated in the aetiology of the AIDS dementia complex (ADC). The rate limiting enzyme for this pathway is indoleamine 2,3- dioxygenase (IDO). We tested the efficacy of different strains of HIV-1 (HIV1-BaL, HIV1-JRFL and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain derived HIV-1 isolates, laboratory adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the high replicating macrophage tropic LA strain, HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day-8 post-infection. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent oxidative tryptophan catabolism in MDM.     

Induction of indolamine 2,3-dioxygenase in primary human macrophages by human immunodeficiency virus type 1 is strain dependent

Increased kynurenine pathway metabolism has been implicated in the etiology of AIDS dementia complex (ADC). The rate-limiting enzyme for this pathway is indolamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of human immunodeficiency virus type 1 (HIV1-BaL, HIV1-JRFL, and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain-derived HIV-1 isolates, laboratory-adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the highly replicating macrophage-tropic LA strain HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day 8 postinfection. Abundant HIV-1 replication did not reduce the ability of exogenous gamma interferon (IFN-gamma) to induce IDO and kynurenine synthesis in HIV-infected MDM. The addition of anti-IFN-gamma antibody to MDM infected with HIV1-JRFL resulted in an absence of detectable IDO protein after 48 h and a decrease of 64% +/- 1% in supernatant kynurenine concentration. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent kynurenine metabolism in MDM. The induction of IDO, while apparently independent of replication capacity, appears to be mediated by a transient production of IFN-gamma in MDM responding to the initial infection with selected strains of HIV-1.     

Human cytomegalovirus infection reduces surface CCR5 expression in human microglial cells,astrocytes and monocyte-derived macrophages

Within the brain, glial cells are target cells for human cytomegalovirus (HCMV) and HIV. We infected cultures of unstimulated human microglial cells and astrocytes of embryonic origin and of monocyte-derived macrophages (MDM) with HCMV strain AD169 and observed down-regulation of the plasma membrane expression of CCR5 in the three cell types, and of CXCR4 and CD4 in microglial cells only. Cells were then coinfected simultaneously or at a 24-h interval with both AD169 and two different HIV-1 monocytotropic strains. HCMV late antigens and HIV-1 tat protein colocalized in the cytoplasm of 5-10% of microglia and MDM. p24 antigen levels decreased 10- to 40-fold in supernatants of MDM and the reduction was greater when HCMV infection was performed 24 h before HIV-1 infection. These data suggest that HCMV-induced reduction in the cell-surface expression of the primary co-receptor of HIV-1 monocytotropic strains may impair the ability of HIV to infect these cells.     

Dysregulation of macrophage-secreted cathepsin B contributes to HIV-1-linked neuronal apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1(ADA) infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages.     

HIV infection of placental macrophages: effect on the secretion of HIV stimulatory cytokines.

Vertical transmission of HIV-1 can occur at three different stages: during gestation, delivery and breast feeding. To determine the role of cytokines in vertical transmission of HIV during gestation, we studied the secretion of IL-1beta, TNF-alpha and IL-6 from in vitro infected and Mock-infected placental macrophages (Hofbauer cells) in comparison to blood monocyte derived macrophages (MDM). Hofbauer cells stimulated with lipopolysaccharide (LPS) secreted lower levels of HIV stimulatory cytokines (6-8 ng/ml) in the supernatants than MDM (26 ng/ml, p<0.005). Cytokine levels in MDM decreased upon HIV infection to 7 ng/ml. IL-6 was the major cytokine produced after LPS stimulation by the two cell populations (p<0.005), being MDM the major cytokine producer. In vitro infection studies with a M-tropic virus (HIV-BaL) indicated that MDM were 10x more susceptible to HIV than placental macrophages (p=0.001). Our results indicate that although macrophages from term placenta secrete lower amount of HIV stimulatory cytokines than MDM, there was no correlation between the levels of cytokines and HIV production by both cells.     

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.     

L-tryptophan-kynurenine pathway metabolites regulate type I IFNs of acute viral myocarditis in mice

The activity of IDO that catalyzes the degradation of tryptophan (Trp) into kynurenine (Kyn) increases after diseases caused by different infectious agents. Previously, we demonstrated that IDO has an important immunomodulatory function in immune-related diseases. However, the pathophysiological role of IDO following acute viral infection is not fully understood. To investigate the role of IDO in the l-Trp-Kyn pathway during acute viral myocarditis, mice were infected with encephalomyocarditis virus, which induces acute myocarditis. We used IDO-deficient (IDO(-/-)) mice and mice treated with 1-methyl-d,l-Trp (1-MT), an inhibitor of IDO, to study the importance of Trp-Kyn pathway metabolites. Postinfection with encephalomyocarditis virus infection, the serum levels of Kyn increased, whereas those of Trp decreased, and IDO activity increased in the spleen and heart. The survival rate of IDO(-/-) or 1-MT-treated mice was significantly greater than that of IDO(+/+) mice. Indeed, the viral load was suppressed in the IDO(-/-) or 1-MT-treated mice. Furthermore, the levels of type I IFNs in IDO(-/-) mice and IDO(-/-) bone marrow-transplanted IDO(+/+) mice were significantly higher than those in IDO(+/+) mice, and treatment of IDO(-/-) mice with Kyn metabolites eliminated the effects of IDO(-/-) on the improved survival rates. These results suggest that IDO has an important role in acute viral myocarditis. Specifically, IDO increases the accumulation of Kyn pathway metabolites, which suppress type I IFNs production and enhance viral replication. We concluded that inhibition of the Trp-Kyn pathway ameliorates acute viral myocarditis.     

Investigation of the kynurenine pathway in Indoleamine 2, 3 dioxygenase deficient mice with inflammatory arthritis

Tryptophan is an essential amino acid involved in the protein synthesis, cognition, and immunity. Oxidative catabolism of tryptophan is executed by the sets of biochemical reactions collectively referred to as the kynurenine pathway. In the immune system, two distinct enzymes, Indoleamne 2,3 dioxygenase 1 (IDO1) and Indoleamine 2, 3 dioxygenase 2 (IDO2) can initiate metabolic flux through the kynurenine pathway. Rheumatoid arthritis is an autoimmune disease driven by the exacerbated immune response towards self antigens and characterized by the chronic inflammatory reaction of the diarthrodial joints. Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis. Using CIA in wild type (WT) and mice deficient with Indoleamine 2,3 dioxygenase (Ido1KO), it was of interest to test the impact of Ido1 deletion on the concentration of tryptophan and its catabolites as well as on mRNA expression for other genes on the kynurenine pathway. Here, when compared with samples taken from naïve WT animals and those with CIA, it was found that only in the inguinal lymph nodes (iLN) taken from Ido1KO mice with CIA tryptophan concentration was significantly increased. In contrast, mRNA expression for Ido2 was decreased in naïve as well as in the diseased iLN taken from Ido1KO mice. Deletion of Ido1 and reduced mRNA expression for Ido2 neither affected the concentration of the downstream metabolites of tryptophan nor mRNA expression for downstream genes on the kynurenine pathway in iLN. Moreover, the concentration of kynurenine in sera of mice with CIA was significantly decreased in Ido1KO mice with arthritis.     

Deficient tryptophan catabolism along the kynurenine pathway reveals that the epididymis is in a unique tolerogenic state

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1(-/-)-deficient mice. In the caput epididymis of Ido1(-/-) animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.     

HIV-1 Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis

HIV-1 Nef is the regulatory protein expressed earliest and most abundantly in the infection cycle. Its expression has been correlated with a plethora of effects detectable either in producer, target, and bystander cells, as well as in the viral particles. Even if the relationship between Nef expression and apoptosis has been already matter of investigation in infected lymphocytes, whose resistance to HIV infection is however limited to few days, this remains to be investigated in cells that in vivo well resist the HIV cytopathic effect. In such an instance, we were interested in establishing whether Nef influences the apoptotic processes in primary human-monocyte-derived macrophages (MDM). High efficiency HIV-1 infection of MDM allowed us to establish that virus-expressed Nef strongly counteracts the HIV-1-induced apoptosis. The Nef mutant analysis suggested that this effect relies on the interaction with different protein partners and cell compartments. We also observed that the Nef protection to the HIV-1-induced apoptosis correlated with the hyper-phosphorylation and consequent inactivation of the pro-apoptotic Bad protein. On the basis of these results, we propose the Nef anti-apoptotic effect as a relevant part of the mechanism of the in vivo establishment of the HIV macrophage reservoirs.     

Candida albicans and HIV-1 Infection

Candida albicans virulence is in part mediated by fibronectin (FN) interaction. We compared the adherence level to FN (using Becton Dickinson FN-coated plates) of several strains of yeast isolated from HIV-1 infected or uninfected subjects affected by candidiasis (30 strains from HIV+ subjects and 18 from HIV- subjects). More adhesive strains were found in HIV+ patients than in HIV- subjects. In particular a mean increase of 120 per cent as regards the total number of adhesive cells and 230 per cent as regards the adhesive cells producing germ tubes was detected in the former group of strains as compared to the latter ( p < 0.001 in both cases). The enhancement of FN expression induced by HIV-1 infection, as we have previously demonstrated, can increase interest in the adherence to FN of C. albicans strains isolated from AIDS-affected patients. Moreover, we also underline the important role played by HIV Nef protein in increasing the C. albicans aggressiveness. In fact a significant inhibitory effect of Nef on the phagocytosis of this yeast by macrophages has been demonstrated and the oxidative processes of these cells seem to be down-regulated by this protein.     

Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease

The kynurenine pathway is the major route for the oxidative degradation of the amino acid tryptophan. Activity of the pathway is involved in several disease conditions, both in the periphery and the central nervous system, including cancer, inflammatory disorders, neurological conditions, psychiatric disorders and neurodegenerative diseases. Three enzymes are now known to catalyze the first and rate-limiting step in the catabolism of tryptophan along this pathway: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO, subsequently named IDO1), both of which have been extensively studied, and a third enzyme, indoleamine 2,3-dioxygenase 2 (IDO2), a relative newcomer to the kynurenine pathway field. The adjuvant chemotherapeutic agent, 1-methyl-d-tryptophan, was intially suggested to target IDO2, implying involvement of IDO2 in tumorigenesis. Subsequently this compound has been suggested to have alternative actions and the physiological and pathophysiological roles of IDO2 are unclear. Targeted genetic interventions and selective inhibitors provide approaches for investigating the biology of IDO2. This review focuses on the current knowledge of IDO2 biology and discusses tools that will assist in further characterizing the enzymes of the kynurenine pathway.     

Macrophages exposed to lymphotropic and monocytotropic HIV induce similar CTL responses despite differences in productive infection

Macrophages are accessory cells that are vulnerable to infection by HIV-1. HTLV-IIIB, a lymphotropic strain of HIV, infects macrophages poorly resulting in either no or low levels of virus expression compared to high levels of productive infection after exposure of macrophages to the monocytotropic HIV strain Ada-M. Whether this results in an impaired ability of HTLV-IIIB-exposed macrophages to initiate protective cytotoxic T lymphocyte (CTL) immune responses against these strains is not well defined. We investigated the ability of monocyte-derived macrophages (MDM) exposed to lymphotropic and monocytotropic HIV strains to initiate primary CTL responses in vitro. MDM exposed to HTLV-IIIB induced a specific primary CTL response that was comparable to MDM exposed to the monocytotropic strain Ada-M despite marked differences in productive HIV infection in MDM between the two strains. CTL generated in this model were MHC-restricted, strain-specific, and CD8+. These data demonstrate that high levels of productive HIV infection in accessory cells are not a prerequisite for the generation of a primary CTL response, suggesting a novel immunologic interaction between MDM and lymphotropic HIV strains.     

Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics.