HIV-1 Tat activates indoleamine 2,3 dioxygenase in murine organotypic hippocampal slice cultures in a p38 mitogen-activated protein kinase-dependent manner

Recombinant Tat 1-72 was provided by Professor Avindra Nath through a contract with the University of Kentucky. Stock solutions of Tat 1-72 were prepared in phosphate buffered saline (PBS) (1ug/ul) and stored at -80°C until use. Recombinant murine IFNγ (cat# 315-05) was from PeproTech, Inc. Heat-inactivated horse serum (cat# SH30074.03), Hank's balanced salt solution (HBSS, cat# SH30030.03) and minimal essential media (MEM) (cat# SH30024.02) were all from Hyclone. Gey's balanced salt solution (GBSS, cat# G9779) was from Sigma, D-glucose (cat# 15023-021) was from GibcoBRL and the kits for enzyme-linked immunosorbent assays (ELISA) were obtained from R&D Systems (Wiesbaden, DE). TRIzol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA). Reagents for RT-PCR were all from Applied Biosystems as follows: high capacity cDNA reverse transcription kit (cat# 4374967); RT-PCR primers for IDO (cat# Mm00492586_m1), TNFα (cat# Mm00443258_m1), IL-6 (cat# Mm00446190_m1), the inducible isoform of nitric oxide synthase (iNOS) (cat# Mm00440485_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat# Mm999999_g1). Antibodies specific for p38 (cat# 9212) and phosphorylated p38 (p- p38, cat# 9211) were purchased from Cell Signaling Biotechnology (Danvers, MA), whereas the secondary horseradish peroxidase (HRP)-linked rabbit anti-mouse antibody (NA934V) was obtained from GE Healthcare Biosciences (Piscataway, NJ). The p38 MAPK inhibitor SB 202190 (cat# 559397) was from EMD Chemicals, Inc. (USA). Protein was measured with a standard Bradford assay kit (cat# 500-0113, 0114, 0115) and Immun-Blot polyvinylidene difluoride (PVDF, cat# 162-0177) membranes were from Bio-RAD (Hercules, CA). ECL Western blotting detection reagents (cat# RPN2106V1, RPN2106V2) were obtained from GE Healthcare Little Chalfont (Bucks, UK).

All animal care and use procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council) and approved by the Institutional Animal Care and Use Committee. Experiments conducted in vivo were performed on 12-week-old male C57BL/6J mice obtained from a colony raised in our laboratory. Mice were individually housed in standard shoebox cages, with wood shavings as litter, in a temperature- (23°C) and humidity- (45-55%) controlled environment with a 12/12 h modified dark-light cycle (lights on 10:00 P.M.-10:00 A.M.). Food and water were available ad libitum.

Mice were surgically implanted with a single guide cannula (Plastics One, Roanoke, VA) directed toward the lateral ventricle, As previously described [10]. The guide cannulas were kept clean and covered using a screw on cannula dummy for mice (Plastics One, Roanoke, VA). Coordinates for placement of the guide cannula were 1.5 mm lateral, 0.6 mm posterior, and 1.3 mm dorsal with respect to bregma. These coordinates placed the guide cannula 1 mm dorsal to the lateral ventricle. Mice were allowed to recover 2 weeks before treatment and initiation of behavioral tests. After recovery, mice were slowly injected over 1 min i.c.v. with phosphate buffered saline (PBS) or Tat (40 ng) in a volume of 1 μl. This dose of Tat was selected on its ability to reliably induce IDO expression in human astrocytes [38].

The forced swim test was conducted at 24 h post i.c.v. injection of Tat for a five-min period and the mice were video recorded for future analysis. Immobility was defined as passive floating behavior or any movement necessary for the mouse to keep its head above water, as described previously [10].

Murine hippocampal slice cultures were prepared using the static interface culture method [22]. Briefly, 6- to 8-day-old C57BL/6J mice were decapitated. The brains and meninges were removed, followed by separation of the hippocampus from both hemispheres. Hippocampi were dissected and transverse slices (350 μm in thickness) were prepared using a McIlwain tissue chopper (Campden Instruments Ltd, UK). Slices were placed for 1 h at 4°C into GBSS supplemented with 2 mg/ml D-glucose and were then transferred onto porous (0.4 μm) transparent membrane inserts (30-mm in diameter; Millipore) with five slices on each insert. Inserts were then placed into six-well culture plates. Each well contained 1.2 ml of nutrient medium composed of 25% heat-inactivated horse serum, 25% HBSS and 50% MEM supplemented with 25 mM D-glucose. Neither antibiotics nor anti-mycotics were used. Plates were maintained in a humidified CO₂ incubator (5% CO₂, 95% atmospheric air) at 37°C. Medium was changed every 2-3 days. The MEM medium was changed so that it contained only 5% horse serum and 25 mM D-glucose on the day that Tat or control medium was added. At various times following addition of Tat, supernatants were collected and stored at -80°C for measurement of cytokines. Slices were washed 3 times with cold PBS and stored at -80°C for isolation of total cellular RNA and for western blotting. Slice viability was evaluated using both propidium iodide (PI) staining and the amount of lactate dehydrogenase (LDH) released into the culture medium by CytoTox⁹⁶ non-radioactive cytotoxicity kit.

Total cellular RNA from the hippocampus and cultured slices was extracted in TRIzol reagent, as previously described [22]. Total mRNA (1-2 μg) was reverse transcribed to cDNA using the high capacity cDNA reverse transcription kit from Ambion. Samples were analyzed in duplicate. Data were analyzed using the comparative threshold cycle method, as described elsewhere (Applied Biosystems user bulletin no.2).

TNFα and IL-6 were measured in OHSCs supernatants with validated specific ELISA assays [22]. Briefly, 100 μl of each sample were added in duplicate to ELISA plates pre-coated with an anti-TNFα or IL-6 capture antibody. Recombinant murine TNFα and IL-6 standards ranged from 0 to 1,000 pg/ml. The lower assay limit of detection was 16 pg/ml. Absorbance was measured on an OPTImax ELISA plate reader. TNFα and IL-6 concentrations are expressed as picograms per milliliter.

Western blotting experiments were conducted as previously described [26], with minor modifications. Briefly, slices were lysed in cold lysis buffer. Equal amounts of protein (40 μg) were separated on 10% polyacrylamide gels. Proteins were then transferred from the gel to PVDF membranes using a Bio-Rad Laboratories Mini Protein 3 system. After treating PVDF membranes with blocking buffer (TBS/0.1% Tween20 (TBST) containing 2% BSA) for 1 h at room temperature, they were incubated overnight at 4 C with blocking buffer containing primary antibodies specific for phosphorylated p38 or p38, (1:1000 dilution). Membranes were washed extensively with TBST and then incubated for 1 h at room temperature with a secondary antibody coupled to horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG antibodies at a dilution of 1:2000 in blocking buffer. Finally, membranes were washed extensively with TBST and developed with an enhanced chemiluminescence ECL Western Blot Detection Reagent. Blots were covered with transparency film and then inserted into a Fujifilm LAS-4000 System Configured for multifunctional analysis (Fujifilm, Life Science, Stamford, USA). Densitometric analysis of autoradiograms was performed using publically available IMAGE-J software from the National Institutes of Health (Bethesda, MD). Densitometric summaries were expressed as ratios of phosphorylated p38 to total p38.

Data were analyzed using a one-way (treatment) or two-way (pretreatment × treatment) ANOVA, followed by a post hoc pairwise multiple comparison using Fischer's LSD test if the interaction was significant. All data are presented as means ± SEM. Differences were considered significant if the probability reached a level of 0.05 or less.

Mice were treated with i.c.v. PBS or Tat (40ng). At 24 h post Tat, sickness and depressive-like behavior were assessed by body weight loss and FST, respectively. Consistent with our previous findings [10], the 24 h changes in body weight did not differ according to treatment (Figure 1A, p > 0.05). Tat-treated mice displayed increased immobility in the FST at 24 h post-treatment compared to control mice (Figure 1B, p < 0.05).

We recently found that central injection of HIV-1 Tat (40 ng) increased brain IDO and cytokine steady-state transcripts at 4 h [10]. Here we determined whether the expression of IDO and cytokines response to Tat occurs in murine OHSCs as it does in the CNS in vivo. Based on the in vivo response to central injection of Tat [10], the time point of 6 h was selected for carrying out dose-response experiments to determine the effect of Tat on cytokine and IDO expression. Slices were exposed to 4, 40 and 400 ng/slice of Tat on day 10 in culture. As shown in Figure 2, Tat significantly increased TNFα, IL-6 and iNOS at the mRNA level (p < 0.05) in a dose-dependent manner, with a maximum at 400 ng/slice. Tat-induced increases in TNFα and IL-6 mRNA expression were paralleled by a concomitant increase in protein production (Figure 2, p < 0.05). Increased iNOS mRNA was not associated with any detectable increase in nitrite levels at the 6 h time point (data not shown). Induction of SERT mRNA reached a maximum at 4 ng/slice (Figure 2, p < 0.05).

For kinetic studies, OHSCs were exposed to 40 ng/slice Tat for 2, 6 and 12 h on day 10 of culture. As shown in Figure 3, the greatest expression of both mRNA and protein for TNFα occurred at 2 h (p < 0.05) and 6 h (p < 0.05), respectively. IL-6 mRNA peaked at 6 h (p < 0.05) and gradually decreased at 12 h. IL-6 concentration in the culture medium increased after 6 h (p < 0.05) and reached a maximum at 12 h (p < 0.05). iNOS and SERT mRNA increased at 6 h (p < 0.05) and peaked at 12 h (p < 0.05), but there was no concomitant increase in nitrite levels at any time point (data not shown). We also used real-time RT-PCR to determine whether Tat induces IDO steady-state transcripts in OHSCs as it does in vivo. To determine an optimal dose for Tat-induced expression of IDO, OHSCs were treated with 4, 40 and 400 ng/slice of Tat for 6 h. As shown in Figure 4A, IDO mRNA expression could be detected at 4 ng/slice Tat and peaked at 40 ng/slice Tat (p < 0.05). Kinetic studies were then carried out in which OHSCs were exposed to Tat (40 ng/slice) for 2, 6 and 12 h. As shown in Figure 4B, IDO mRNA could not be detected in OHSCs prior to addition of Tat (40 amplification cycles). However, IDO expression was significantly induced by Tat at 6 h (p < 0.05) with no further increase at 12 h. This effect was similar in intensity to that of exogenous IFNγ (10 ng/ml, Figure 4C, p < 0.05). Moreover, pretreatment of OHSCs with the same dose of IFNγ 24 h before Tat markedly amplified IDO responses to Tat (Figure 4C, p < 0.01). However, the Tat-induced expression of IDO did not require endogenous synthesis of IFNγ because no IFNγ mRNA could be detected at 6 h in Tat-stimulated slices (40 amplification cycles, data not shown).

In accordance with the results of others on the effects of Tat on p38 MAPK in various cell types [32‐36], we confirmed that HIV-1 Tat can activate p38 MAPK in OHSCs. Slices were treated with Tat for 15, 30, 60 and 120 min respectively and the lysates were analyzed for phospho-p38 activity by Western blot analysis. We found that Tat induced significant phosphorylation of p38 MAPK as early as 15 min (Figure 5A, p < 0.05) with a maximum at 60 min (Figure 5A, p < 0.05).

To determine whether p38 MAPK is involved in Tat-induced IDO expression, the p38 MAPK inhibitor SB 202190 was employed. We previously established that 30 μM SB 202190 significantly suppresses cytokine expression at both the mRNA and protein levels in response to LPS stimulation (data not shown). The dose of 30 uM was therefore selected for further experiments on Tat-induced IDO expression. Slices were pretreated with SB 202190 (30 μM) for 30 min before stimulation with Tat for another 6 h. SB 202190 abrogated the Tat-induced expression of IDO, IL-6, iNOS and SERT transcripts, although this effect was only partial for TNFα mRNA (Figure 5B, p < 0.01). SB 202190 also fully blocked the Tat-induced release of cytokine proteins in the slice supernatants (Figure 5B, p < 0.01). Importantly, neither Tat nor SB 202190 affected viability of the cells, as determined by measuring both PI staining and release of lactate dehydrogenase into the culture medium (data not shown). These data clearly demonstrate that the p38 MAPK signaling pathway is necessary for the Tat-induced expression of both proinflammatory cytokines and IDO.