HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR

HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49–57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

maintained in DMEM medium with high glucose (Vitrocell Embriolife, Campinas, SP, Brazil) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). THP-1 cells were differentiated into macrophages by incubating with 40 ng/mL of PMA for 3 days. Then, the cells were washed 3 times with PBS and incubated with fresh medium for an additional 3 days. RAW 264.7 cells expressing either empty vector (RAW-WT-Bla cells) or a dominant-negative PKR K296R (RAW-DN-PKR cells) were provided by Dr. Aristóbolo Silva (Federal University of Minas Gerais, Brazil). Monocyte-derived macrophages were obtained from peripheral blood mononuclear cells (PBMCs) isolated from buffy coat preparations of human healthy blood donors as previously described 21 . Thioglycolate-elicited peritoneal macrophages from wild-type or PKR-knockout 129 Sv/Ev mice were obtained by injecting 8 mL of serum-free DMEM into the peritoneal cavity. After five days, the cells were washed in PBS one time and then plated in in DMEM medium supplemented with 10% FBS on glass coverslips at a density of 2 × 10 5 /well in 24-well polystyrene plates for subsequent Leishmania infection assays. All experimental procedures involving human cells were approved by the Oswaldo Cruz Foundation/Fiocruz Research Ethics Committee (Rio de Janeiro, RJ, Brazil), under the number 397-07. The experimental protocols using mice were approved by the Federal University of Rio de Janeiro Committee for Animal Use (permit numbers: IMPPG 024 and IBCCF171. Parasites, culture conditions and infection. Leishmania (L.) amazonensis (WHOM/BR/75/ Josefa) was used in this study. Promastigote forms were grown at 26 °C in Schneider's Insect Medium (Sigma-Aldrich) with 10% fetal bovine serum and were used at the stationary growth phase (5-6 days). Macrophages were infected with Leishmania promastigotes with a parasite: cell ratio of 5:1 at 37 °C. In some experiments, THP-1 cells were treated with 300 nM of the PKR inhibitor. Recombinant HIV-1 Tat (100 ng/mL) was added to the cell cultures after Leishmania infection (the use of this concentration was based on our previous published work 21 ). Infected macrophages were counted by light microscopy to assess the infection index, which was calculated by multiplying the percentage of infected macrophages by the average number of parasites per macrophage in Giemsa-stained slides. Promastigote production derived from infected cells was evaluated as follows: after 3 days of infection, non-adherent cells were removed and the adherent cells were washed 3 times with 1x PBS. The adherent cells were added to the wells in 0.5 mL of Schneider medium containing 20% FBS. After five to seven days at 26 °C, the growth of extracellular motile promastigotes originating from the infected macrophages was assessed by counting in a Neubauer chamber.
Safety of Leishmania treatment with Tat and PKR inhibitor. Leishmania promastigotes were treated with Tat or oxidized-Tat (both at 100 ng/mL), or with 300 nM of the PKR-inhibitor for 1 h. Then, the promastigotes were washed with PBS and incubated with peritoneal macrophages (obtained as described above) at a 1:5 parasite: cell ratio for 1 h or 48 h at 35 °C in 5% CO 2. The infection index was calculated as described above and used to determine parasite survival.

HIV-1 isolate and co-infection. THP-1 cells were infected with the monocytotropic CCR5-dependent
HIV-1 isolate Ba-L using viral suspensions containing 5-10 ng/mL of HIV-1 p24 antigen. Excess virus was washed out after 24 hours and then fresh medium was added back. The cells were maintained under standard culture conditions for 10 days. Subsequently, macrophages were infected with L. amazonensis promastigotes with a parasite: cell ratio of 5:1 at 37 °C overnight. Non-internalized promastigotes were washed out, fresh medium was added, and the cultures were maintained at 37 °C in 5% CO2 for an additional 72 hours for counting by light microscopy to estimate the infection index. For HIV-1 Tat neutralization, rabbit anti-HIV-1 Tat or the IgG isotype control were employed as follows: the supernatant of HIV-1 infected macrophages was removed and treated with anti-Tat, the IGg1 isotype or iPKR. Fresh medium was added and the promastigotes were allowed to interact with the cultures for one hour. After this period of time, the treated supernatant was added to the infected macrophages and the infection index was estimated as described above.
Nitric Oxide Production. The Griess reaction was employed to analyze the nitrite (NO2 − ) content as an indicator of NO production in the supernatant of RAW 264.7 cell lines accordingly the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). The reaction was read at 540 nm and the NO2-concentration was determined with reference to a standard curve generated using sodium nitrite. The results were expressed as micromolar concentrations of nitrite.
Quantitative Real Time RT-PCR. Total RNA from THP-1, wild-type RAW 264.7 and DN-PKR cells (1 × 10 6 cells) was extracted with the Invitrap ® Spin Cell RNA mini kit (STRACTEC Molecular GmbH, Berlin, Germany). A 1 μ g aliquot was reverse transcribed to first-strand cDNA with ImProm-II (Promega) and the oligo(dT) primer according to the manufacturer's instructions. The DNA sequences of the primers used in this work are: Hu-PKR-F: 5'-GACCTTCCTGACATGAAAGAA-3′ and Hu-PKR-R: 5′ -AACTATTTTTGCTGTTCTCAGG-3′ ; GAPDH-F: 5′ -TGCACCACCAACTGCTTAGC-3′ and GAP DH-R: 5′ -GGCATGGACTGTGGTCATGAG-3′ ; Mu-iNOS-F: 5′ -CAGCTGGGCTGTACAAACCTT-3′ and Mu-iNOS-R: 5′ -CATTGGAAGTGAAGCGTTTCG-3′ ; and Hu-IL10-F: 5′ -AGAACCTGAAG ACCCTCAGGC -3′ and Hu-IL10-R: 5′ -CCACGGCCTTGCTCTTGTT-3′ . Amplicon specificity was carefully verified by the presence of a single melting temperature peak in the dissociation curves run after real-time RT-PCR, and the detection of a single band of the expected size was examined by electrophoresis. Real-time quantitative RT-PCR (qRT-PCR) was performed via the Applied Biosystems StepOne TM detection system (Applied Biosystems) using the GoTaq ® qPCR Master Mix (Promega Corp., Madison, WI, USA). All qRT-PCR experiments were performed at least 3 times. qRT-PCR data from the experiments were normalized using GAPDH as an endogenous control. All expression ratios were computed via the analysis of the relative gene expression using the Δ Δ Ct method through StepOne software version 2.0 (Applied Biosystems). Plasmids for Tat overexpression. Cloned plasmids with the Tat gene isoforms were kindly provided by Dr. Mauro Giacca (ICGEB -International Centre for Genetic Engineering and Biotechnology, Italy). The group provided two isoforms: Tat-86 wild-type and Tat86 R(49-57)A, which is a basic-domain mutant 19 . The sequences were amplified by PCR and subcloned into pEGFP-N2 (ClonTech) between the HindIII and EcoRI restriction sites. The constructs were transfected into THP-1 cells (2 × 10 6 ) using 500 ng of DNA with the Nucleofector TM Technology (Lonza, Basel, Switzerland) according to the manufacturer's instructions.
Statistical analyses. Data were analyzed by One-way ANOVA for independent samples using Prism

Tat enhances PKR expression in macrophages infected with L. amazonensis. L. amazonensis
induces the expression of PKR and triggers PKR signaling in macrophages 6,10 . Because PKR signaling plays a pivotal role in the host macrophage-Leishmania interaction, we investigated whether the HIV-1 Tat protein would enhance the expression and the levels of activated PKR in Leishmania-infected macrophages. We used a luciferase gene reporter construct containing inducible elements of the PKR promoter to test the role of Tat in Leishmania-infected macrophages. As observed in Fig. 1A, the addition of Tat increased the activity of the PKR promoter and enhanced the luciferase levels induced by parasite infection. Accordingly, PKR expression was highly increased in infected macrophages treated with Tat ( Fig. 1B,C). The pre-incubation of HIV-1 Tat with Leishmania did not affect the parasite association with macrophages or the parasite load 48 hours post-infection, thereby ruling out a direct effect of Tat on the parasite (Supplementary Figure 1). Importantly, PKR underwent an increase in phosphorylation in Leishmania-infected cells treated with Tat (Fig. 1D), as revealed by the use of anti-pPKR (Thr451). Therefore, we investigated whether the main PKR substrate, the initiation factor eIF2α , was phosphorylated. Figure 1E shows that eIF2α was phosphorylated in infected macrophages. However, the addition of Tat remarkably reduced eIF2α phosphorylation, suggesting that Tat may be a PKR substrate and compete with eIF2α , as described elsewhere 14 . Taken together, these results indicate that HIV-1Tat is able to induce an increase in PKR levels and phosphorylation upon Leishmania infection.
PKR is critical for the Tat-induced enhancement of intracellular Leishmania growth. The above results prompted us to investigate the impact of the Tat-mediated enhancement of PKR activation on Leishmania intracellular growth. To this end, we used a RAW 264.7 cell line stably expressing a dominant-negative (DN)-PKR protein 24 or PKR-KO macrophages. As depicted in Fig. 2A,B, Tat-induced growth of Leishmania was impaired in PKR-KO macrophages and DN-PKR RAW 264.7 cell line, thus suggesting that PKR is crucial for the Tat effect on intracellular parasite replication. To corroborate these results, we treated the Leishmania-infected THP-1 macrophage cell line or primary human macrophages with the PKR inhibitor CAS 608512-97-6 in the presence of Tat. Accordingly, PKR inhibition strongly reduced the effect of Tat on parasite growth (Fig. 2C,D). Moreover, the pre-incubation of iPKR with Leishmania did not affect the parasite association with macrophages or the parasite load 48 hours post-infection, thereby ruling out a direct effect of iPKR on the parasite (Supplementary Figure 1). Next, we addressed the effect of Tat in the context of HIV-1 and Leishmania co-infection. As expected, HIV-1 infection stimulated parasite growth, and additional treatment with Tat almost doubled parasite replication (Fig. 2E). Then, we investigated whether cells exposed to both HIV-1 and L. amazonensis exhibited enhanced parasite growth in a TAT/PKR-dependent manner. Figure 2F shows that PKR inhibition reduced parasite growth in HIV-1-/Leishmania co-infected cells. The same effect was observed when neutralizing anti-Tat antibody was added to the cultures. Moreover, the parasite load was further reduced when human macrophages were treated with iPKR and anti-Tat was added to the infected cells. Taken together, this set of results strongly suggests that Tat requires host cell PKR signaling to promote the growth of amastigotes in macrophages.
Scientific RepoRts | 5:16777 | DOI: 10.1038/srep16777 L. amazonensis infection down regulates Tat-induced NF-κB activation downstream of PKR signaling. The transcription factor NF-κ B is activated in viral and parasitic infections 25 . Briefly, the heterodimer RelA/p50 is translocated to the nuclei of infected cells and regulates the expression of a number of cytokines 26 . RelA/p50 is also important for HIV-1 LTR expression and may be induced by bacterial co-infections 27 or by HIV-1-encoded proteins such as Tat 28 . We investigated the activation of NF-κ B in L. amazonensis-infected macrophages treated with Tat and the PKR inhibitor through gene reporter assays (Fig. 3A). The NF-κ B reporter construct was induced by Tat, and the inhibition of PKR partially prevented this effect. Importantly, Tat treatment increased NF-κ B activation in uninfected cells, while Leishmania infection diminished this activation. These results were corroborated by employing the DN-PKR-RAW 264.7 cell line, thereby strengthening the hypothesis that Tat-mediated NF-κ B activation was dependent on PKR signaling and that Leishmania infection reduced this effect (Fig. 3B). Next, we investigated the nuclear levels of RelA (p65) induced by Tat and the dependence of this phenomenon on PKR. Figure 3C shows that Tat increased the nuclear levels of RelA and that PKR inhibition prevented this effect. Taken together, these observations corroborate previous results on the role of Tat in NF-κ B activation 29 and demonstrate the importance of PKR signaling in this event. Importantly, L. amazonensis infection per se did not induce the canonical NF-κ B p65/50 dimer, as previously described 30 .

Leishmania infection reduces PKR-dependent Tat-induced iNOS expression.
The production of nitric oxide (NO) is associated with the activation of macrophages and can reduce the number of Leishmania amastigotes 31 . Because HIV-1 Tat has been demonstrated to induce iNOS 32 , we investigated whether L. amazonensis interferes with iNOS expression and NO production in the context of the Tat-PKR axis. We found that Tat induced iNOS expression in a PKR-dependent fashion (Fig. 4A). The presence of Leishmania reduced this effect, suggesting that Leishmania impaired the induction of iNOS by Tat. Accordingly, NO production followed the same pattern (Fig. 4B). These results were corroborated by immunoblot assays. Figure 4C clearly shows that Tat induced iNOS expression via PKR and that this effect was reduced in the presence of the parasite. To evaluate the effect of Tat on the NF-κ B/STAT1 regulatory elements present in the iNOS promoter 33 , we performed luciferase reporter assays. Sole infection by L. amazonensis did not induce the constructs carrying either the NF-κ B/STAT1 or STAT1 regulatory element (Fig. 4D,E, respectively). Importantly, Tat treatment resulted in the activation of both reporter constructs, but Leishmania infection reduced this effect on the NF-κ B/STAT1 construct. Based on these results, we suggest that L. amazonensis reduced the expression of iNOS induced by HIV1 Tat.

The Tat basic domain (49-57) is required for PKR activation and parasite growth enhancement in macrophages.
Tat binds to PKR through a cognate basic domain (49-57 aa) 16 . To address whether the Tat effect on the modulation of parasite infection requires the Tat-basic domain, we used a Tat mutant construct with the arginine (R) residues replaced by alanine (A) (Tat86-R(49-57)A) to evaluate whether both Tat constructs could activate the HIV-1-LTR reporter construct in THP-1 macrophages. As predicted, Tat86-R (49-57)A could not induce HIV-1 LTR activity (Fig. 5A), while Tat86-WT strongly stimulated the LTR regulatory elements. Importantly, the pharmacological inhibition of PKR decreased LTR induction. These results prompted us to evaluate the effect of Tat86-R(49-57)A on intracellular parasite growth. As shown in Fig. 5B,C, Tat86-R(49-57)A did not promote the enhancement of the parasite load. Then, we analyzed the role of Tat in PKR activation in macrophages infected with Leishmania infection. As observed in Fig. 5D, the expression of Tat86-R(49-57)A in Leishmania-infected macrophages did not activate PKR, while the Tat86-WT isoform potentiated pPKR levels. Taken together, our results indicate that Tat favors L. amazonensis growth in a manner that is dependent on the Tat basic region and is possibly mediated through PKR.
Tat favors L. amazonensis growth through PKR-dependent IL-10 expression. We previously reported that PKR activation induces IL-10, which in turn favors L. amazonensis infection 6 . Because HIV-1 Tat may also induce IL-10 expression, we studied the expression of IL-10 in infected macrophages treated with Tat and tested the role of PKR on IL-10 induction. Figure 6A shows that Tat potentiates the ability of L. amazonensis to induce IL-10 expression in macrophages in a PKR-dependent fashion. Similar results were obtained in infected DN-PKR RAW 264.7 macrophages (Supplementary Figure 2). Next, we tested the requirement for the Tat basic domain for IL-10 induction in infected macrophages. Our results showed that the Tat86 construction with the mutated basic domain was unable to induce IL-10 and, as predicted, it could not synergize with the L. amazonensis infection (Fig. 6B). To further characterize the effect of the axis formed by Tat/IL-10 on the exacerbation of parasite growth, we assessed the parasite growth in infected cells treated with Tat in the presence of an anti-IL-10 receptor neutralizing antibody. Blockage of the IL-10 receptor abolished the effect of Tat on parasite growth (Fig. 6C). Moreover, the enhancement of Leishmania growth by Tat86-WT was also dependent on IL-10, whereas the mutant construct Tat86-R (49-57)A could not exacerbate parasite growth (Fig. 6D).

Discussion
The soluble HIV-1 Tat protein can be found in in the serum of HIV-1 infected patients as well as in the tissues 34 . Once in the extracellular milieu, Tat can be taken up by bystander host cells 35 . Inside the cells, Tat is able to interact and activate signaling pathways 36 , which can result in the induction of cytokines such as IL-10 13 and TGF-β 37 . Tat was previously reported to induce the uptake of Leishmania parasites 38 . We also demonstrated that Tat increased the intracellular growth of Leishmania amazonensis and revealed the importance of secreted factors in the Leishmania-exacerbation effect mediated by Tat 21 . However, neutralization of two soluble factors (Prostaglandin E2 and TGF-β ) did not completely abolish the up-modulating effect of Tat on Leishmania 21 , suggesting that other mediators (or even interference with a cellular signaling pathway) could participate together with these secreted molecules. The dsRNA-activated kinase PKR can be targeted and activated by Tat 14,17 . Importantly, previous work demonstrated a pivotal role for PKR in the growth of intracellular parasites 6,10,11 . These observations strengthened our hypothesis that Tat could also affect the cell signaling cascade without requiring prior cytokine secretion. In this work, we tested the hypothesis that Tat could modulate the growth of intracellular parasites in infected macrophages via PKR signaling and IL-10 expression.
Our results clearly showed that Tat potentiated PKR activation resulting from Leishmania infection. The PKR binding sequence of the main PKR substrate (the translation initiation factor eIF2α ) shares amino acid sequence similarities with the phosphorylation region of HIV-1 Tat. Our results confirmed that L. amazonensis infection leads to PKR activation and the phosphorylation of eIF2α . This effect was prevented by the addition of HIV-1 Tat. HIV-1Tat 86 can be phosphorylated by PKR, thus competing with the PKR substrate eIF2α 14 . Importantly, the PKR promoter construct was activated by Tat treatment or Leishmania infection alone, and the association of both conditions increased the stimulation of the PKR promoter, probably through the induction of ISRE elements present in the construct. We cannot rule out a direct effect of HIV-1 Tat on the activation of these responsive elements. However, we observed the induction of IFN1β expression in Tat-treated macrophages (data not shown), which could lead to the activation of the PKR promoter.
As expected, based on our previous studies 21 , HIV-1 Tat enhanced amastigote growth in macrophages and now we show that this phenomenon requires PKR signaling. Importantly, the neutralization of HIV-1 Tat produced by HIV-1-infected macrophages reduced the parasite load, and this effect was even more accentuated when anti-HIV-1 Tat was added to iPKR-treated Leishmania-infected macrophages. PKR and Tat act together to induce IL-10 expression 39 and our results reveal an important aspect of Leishmania and HIV-1 co-infection: HIV-1 Tat protein is able to induce IL-10 secretion in Leishmania-infected macrophages through PKR activation. This conclusion is supported by our results showing that IL-10 expression was highly induced in Tat-treated Leishmania-infected macrophages, while there was reduced expression of IL-10 in the infected DN-PKR RAW 264.7 cell line and human macrophages treated with iPKR. The blockage of the IL-10 receptor completely abrogated the Tat effect in promoting the intracellular growth of Leishmania, thus corroborating the notion that IL-10 is an essential effector of the Tat-PKR axis during co-infection. Moreover, we expressed in infected macrophages a Tat mutant construct in which the basic region was altered, which resulted in a protein that was unable to interact with PKR 19 . The expression of Tat86-R (49-57)A in infected macrophages did not enhance parasite growth or up-modulate IL-10 expression and PKR activation. We have not pursued co-immunoprecipitation Non-internalized promastigotes were washed out and fresh medium was added, and then the cultures were treated for an additional five hours with Tat (100 ng/mL). Western blot analysis of iNOS protein expression was performed. RAW 264.7 cell lines were transiently transfected with the 3XNS-Luc (D) or 3XS-Luc (E) vectors. Then, 24 h post-transfection the cells were infected with L. amazonensis for 18 h and/or treated with Tat (100 ng/mL). After twenty-four hours, the whole cell lysates were analyzed for luciferase activity. The results are representative of three independent experiments. *P < 0.05.  studies to verify the direct binding of HIV-1 Tat 86 to PKR. However, our results are in accordance with previous reports that showed and mapped the minimum sequences involved in the direct interaction of Tat with PKR.
Considering the cascade of signals, HIV-1 Tat could induce IL-10 production even from non-infected cells, and this secreted IL-10 could act in nearby Leishmania-infected cells 40 . HIV-1 Tat can activate the canonical NF-κ B pathway, leading to the nuclear translocation of the RelA/p50 heterodimer 41 , which may contribute to the expression of pro-inflammatory cytokines and iNOS. Our results show that Tat indeed promotes iNOS expression and NO production. However, both effects were reduced in Leishmania-infected macrophages. Accordingly, L. amazonensis subverts canonical NF-κ B activation in macrophages and promotes the formation of the repressor homodimer p50/50 30 , which silences the nk regulatory elements observed in the iNOS promoter. These results show that L. amazonensis interferes with cell host signaling induced by HIV-1 Tat, thereby reducing iNOS production and modulating NF-κ B dimer activation. Taken together, we envisage a scenario in which released Tat enters the cell, interacts with PKR, and regulates its activation. This complex promotes nuclear translocation of NF-κ B and IL-10 induction. Despite the fact that PKR activation could induce a harmful environment for Leishmania survival, the presence of the parasite overrides the effect of PKR action on iNOS expression, thereby permitting parasite survival and growth. Altogether, our data support the notion that Tat plays a pivotal role in favoring L. amazonensis infection through PKR signaling and IL-10 expression. Our results unveil the basic molecular mechanisms by which Leishmania-HIV-1 co-infection may result in the clinical aggravation of leishmaniasis symptoms, and may contribute to the development of novel treatment procedures.