Results of the present experiments establish that HIV-1 Tat induces depressive-like behavior in vivo and induces production of proinflammatory cytokine in vitro in murine OHSCs and increases expression of IDO and SERT. These changes are dependent on activation of the p38 MAPK signaling pathway.
Our in vivo data show that Tat increases the duration of immobility in the forced swim test in mice without inducing any sickness, as measured by the lack of body weight loss. These data are in agreement with already published findings from our group on depressogenic activity of Tat . The mechanisms of the depressogenic activity of Tat were further investigated in vitro. Compared to primary cultures of brain cells, OHSCs have the advantage of preserving the cellular and connective organization as well as several fundamental in vivo-like characteristics such as glial-neuronal interactions [39, 40]. Although this preparation has previously been used for studying the detrimental effects of proinflammatory cytokines on long-term potentiation in the rat system [41, 42], OHSCs have rarely been used for the investigation of neuroimmune interactions. Our previous experiments demonstrated that long term culture of OHSCs can be reliably used to study neuroimmune mechanisms of induction of IDO . We therefore employed OHSCs in the present experiments to study the mechanisms involved in the expression of IDO by Tat.
Depression is an important comorbid condition of HIV infection. We previously demonstrated that IDO, the first and rate-limiting enzyme in the synthesis of KYN from the precursor of Trp, is both sufficient and necessary to mediate depressive-like behavior in response to either acute or chronic activation of the immune system in mice [43, 44]. Moreover, the increase in brain IDO activity is invariably preceded by enhanced expression of IDO mRNA, which can therefore be used as a surrogate marker of IDO activation [26, 43, 44]. Therefore, in the present study, we examined the effects of Tat on IDO mRNA in OHSCs. As shown in Figure 2, HIV-1 Tat protein significantly up-regulates IDO mRNA expression in OHSCs. These results are consistent with previous studies that describe Tat-mediated induction of the IDO expression in other types of cells [19–21]. These in vitro data are also in accordance with our in vivo results showing that Tat induced depressive-like behavior is associated with increased expression of IDO in the brain . Taken together, these data provide the first evidence to indicate that activation of IDO in response to Tat stimulation in the brain could be a key event in the switch from sickness to depressive-like behavior.
IFNγ is considered to be the prototypical inducer of IDO in a variety of cells  as well as in clinical situations in which inflammation-associated depression occurs . Additionally, IFNγ is increased in the brain during HIV infection  and synergizes with Tat to play a critical role in the pathogenesis of HAD . It is well documented that monocytes/macrophages/microglia can produce IFNγ [46–48]. However, IDO expression appears to be up-regulated in an IFNγ-independent manner in HIV-infected human macrophages . IFNγ protein remained undetectable in HIV-infected human macrophage supernatants while IDO expression increased . Our data indicate that IDO induction by Tat does not necessarily require synthesis of IFNγ because no IFNγ transcripts could be detected at 6 h. These observations are in accordance with the results obtained by Boasso et al. . Boasso et al. reported that blockade of either type I or type II IFNs by antibodies was ineffective in preventing the induction of IDO in human peripheral blood mononuclear cells exposed to R5- or X4-trophic HIV. Therefore, our data extend the developing concept that IDO can be induced by Tat in an IFNγ-independent mechanism.
Addition of Tat to OHSCs induced the synthesis and release of proinflammatory cytokines. These cytokines are well known to stimulate HIV-1 replication and contribute to HIV pathogenesis [50, 51]. Increased expression of proinflammatory cytokines in the brain is observed in HIV-1 infected patients . Tat also can up-regulate cytokine expression, such as IL-1β and TNFα, in peripheral blood macrophages, CNS-derived cell lines and primary astrocytes, microglia [53, 54] and human monocytes . The large increase in proinflammatory cytokine expression could mediate IDO induction by Tat. Both in vivo and in vitro studies have shown that IDO induction is associated to IL-1β. For instance, pretreatment in vivo with the anti-inflammatory tetracycline derivative minocycline attenuates LPS-induced expression of brain IL-1β, indicating that IL-1β probably participates in LPS-induced expression of brain IDO . A synergistic activation of IDO by IL-1β, TNF-α and IL-6 has been reported in human monocytic THP-1 cells exposed to LPS . We observed that IL-1β mRNA can be induced by Tat in OHSCs (data not shown). However, this induction does not lead to release of IL-1β protein if exogenous ATP is not added to the culture. This is because ATP is necessary for processing and release of the mature IL-1β protein . In the absence of IL-1β, the most likely mediators of IFNγ-independent IDO induction in OHSCs are TNF-α and IL-6. IDO induction has been shown to be mediated mainly by TNF-α, but not by IL-6 in human monocytic THP-1 cell cultures exposed to immune stimulation . However, IL-6 can synergize with TNF-α to increase IDO activity . The exact cytokine signaling pathways that are predominantly involved in the production of IDO in response to Tat in OHSCs remain to be elucidated.
In addition to IDO induction by Tat, we observed a robust induction of SERT mRNA in response to Tat. This finding indicates that OHSCs can serve as a reliable in vitro model for investigating the possible contribution of serotonin re-uptake mechanisms in comorbid depressive disorders in HIV-infected patients. Whether this change at the mRNA level translates into functional changes in SERT remains to be established. This is an important perspective since differences in the expression and function of SERT are well known to affect many human and mouse quantitative traits, including anxiety- and depression-related behaviors .
To elucidate the signaling pathways that mediate Tat-induced IDO expression, we examined whether Tat contributes to overexpression of IDO by activating p38 MAPK. This signaling pathway has been shown to be required for IDO expression in THP-1 cells following LPS stimulation . Moreover, HIV-1 Tat protein has been reported to activate p38 MAPK in a variety of cells, including monoctyes [32, 33], macrophages , astrocytes  and the human THP-1 cell line . We therefore tested the possibility that Tat-induced p38 promotes expression of IDO in OHSCs. SB 202190, a highly selective, potent and cell permeable inhibitor of p38 MAPK , was employed to inhibit p38 activation. It binds within the ATP pocket of the active kinase with a Kd of 38 nM, as measured in recombinant human p38, and selectively inhibits both the p38α and β isoforms. We observed that SB 202190 significantly inhibited Tat-induction of IDO in OHSCs, which is consistent with the possibility that p38 may be involved in the development of Tat-induced depressive-like behavior.
The demonstration of an inhibitory effect of SB 202190 on Tat-induced changes in OHSCs does not mean p38 MAPK signaling is directly responsible for these effects. Activation of p38 MAPK could act indirectly via NFkB activation [60, 61] or AP-1 activity at both the transcriptional and post-transcriptional levels . The human IDO promoter region contains multiple AP-1 and NFkB sites . Therefore, the possibility that other transcription factors are involved in Tat-induced IDO expression cannot be dismissed. However, our data clearly demonstrate that Tat-induced IDO expression in OHSCs is mediated, at least in part, through a p38-dependent mechanism.
In conclusion, results of the present studies demonstrate that p38 MAPK is potently involved in HIV-1 Tat-induced IDO expression. These studies provide further evidence for targeting the brain IDO and p38 MAPK signaling pathway in the treatment of depressive disorders associated with HIV infection.