Miltefosine Represses HIV-1 Replication in Human Dendritic Cell/T-Cell Cocultures Partially by Inducing Secretion of Type-I Interferon

Virology 432 (2012) 271–276

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Virology

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Miltefosine represses HIV-1 replication in human dendritic cell/T-cell cocultures partially by inducing secretion of type-I interferon

Ravendra Garg a, Michel J. Tremblay a,b,n a Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Que´bec, CHUL, Canada b De´partement de Microbiologie-Infectiologie et Immunologie, Faculte´ de me´decine, Universite´ Laval, Que´bec, Canada article info abstract

Article history: Miltefosine (Milt) was originally synthesized as an antineoplastic agent but this phospholipid drug is Received 27 March 2012 now clinically used as an antiprotozoal compound. We demonstrate here that Milt reduces replication Returned to author for revisions of HIV-1 in cocultures of human dendritic cells (DCs) and CD4 þ T cells. This phenomenon is due to a 7 May 2012 rapid secretion of soluble factors by DCs. We present evidence that the Milt-mediated repression in Accepted 24 May 2012 virus production is associated with induction of type-I interferon (IFN) in DCs. The Milt-dependent Available online 15 June 2012 diminution in HIV-1 production was not totally abrogated by B18R, a vaccinia virus-encoded Keywords: neutralizing type-I IFN receptor, which suggests the involvement of another yet to be identiﬁed HIV-1 soluble factor. Altogether, these results suggest that a therapy with Milt when used to control AIDS protozoan infections in individuals also carrying HIV-1 might also help to limit viral load. Additional Miltefosine studies are warranted to estimate the exact therapeutic potential of Milt as an anti-HIV-1 agent. T cells and dendritic cells & 2012 Elsevier Inc. All rights reserved.

Introduction treatment as comparison to seronegative patients. First-line drugs for the treatment of VL in both HIV-1-infected and non-infected The emergence of the acquired immunodeficiency syndrome patients are the lipid formulations of amphotericin B, pentavalent (AIDS) epidemic in the early 1980s has changed dramatically the antimonials and miltefosine (Alvar et al., 2008). However, several disease pattern caused by Leishmania parasites. For example, studies in co-infected individuals have shown only moderate species that traditionally caused one type of disease have been success rates with regard to initial cure and relapses. shown to be capable of causing other types of diseases. The Miltefosine (Milt) is a phosphorylcholine ester of hexadecanol protozoan parasite Leishmania is highly prevalent in many areas which is acting as a membrane-active synthetic ether-lipid of the world and in particular visceral leishmaniasis (VL) is now analog. This compound was originally discovered and synthesized becoming of higher clinical importance in individuals infected as an antineoplastic, particularly active against breast cancer with human immunodeficiency virus type-1 (HIV-1) because the metastases. Milt has been recently found to be highly active distribution of both human pathogens overlaps in various parts of against Leishmania species both in in vitro and in vivo studies the world (Alvar et al., 2008). Furthermore, leishmaniasis’ classi- (Croft et al., 1987; Murray and Delph-Etienne, 2000; Sundar et al., fication as a childhood disease has been drastically altered due to 1998). The expanded use of Milt for treating VL is due to its HIV-1 and Leishmania co-infections. There is also a growing convenient oral administration route, the increasing leishmanial consensus that VL has emerged as an important opportunistic resistance to antimonials and the unaffordable cost of liposomal infection in AIDS patients (Alvar et al., 1997; Tremblayet al., 1996; amphotericin B. It has been reported that Milt is effective at Wolday et al., 1999). controlling Leishmania parasitemia in both immunodeficient The major challenge in dually infected patients is the admin- BALB/c and nude mice (Le Fichoux et al., 1998; Murray and istration of a successful treatment regimen during the acute Delph-Etienne, 2000). This pre-clinical information has supported phase of the disease. The situation is further complicated by the its general use in individuals infected also with HIV-1 because fact that HIV-1-positive individuals show higher rates of drug these are known to be immunodeficient. Indeed, Milt has been toxicity and relapses, as well as lower cure rates with initial used for the treatment of VL in AIDS patients and has also been employed with some success in cases with recurrent leishmaniasis in HIV-1-infected individuals (Rihl et al., 2006; Schraner et al., n Correspondence to: Centre de Recherche en Infectiologie, RC709, Centre 2005). Interestingly, a number of reports have demonstrated that Hospitalier Universitaire de Que´bec, CHUL, 2705 Boul. Laurier, Que´bec (QC), Canada G1V 4G2. Milt exerts significant immunomodulatory properties in different E-mail address: [email protected] (M.J. Tremblay). cell types (Eue, 2002; Hochhuth et al., 1992).

It is now well accepted that HIV-1 mainly infects CD4þ T cells have no effect on HIV-1 replication in CD4þ T cells in absence of but can also replicate in other cell subpopulations such as iDCs (data not shown). The colorimetric MTS cell proliferation macrophages and dendritic cells (DCs) (Coleman and Wu, 2009; assay revealed that the observed reduction in virus production is Wu and KewalRamani, 2006). DCs are potent and versatile not due to cell toxicity since cellular viability remains the same in antigen-presenting cells and recent evidence suggests that they the presence of all tested concentrations of Milt (data not shown). play a pivotal role in establishment and dissemination of HIV-1 Moreover, similar observations were made when using a CFSE- infection. The infection process seems to be facilitated by virtue of based viability and proliferation test (data not shown). DCs being the initial target cell type of HIV-1 and of their unique Milt represses virus replication in CD4þ T cells. We next tried to capacity to migrate from virus entry sites in the periphery to the T shed light on the mechanism(s) by which Milt can reduce HIV-1 cell-rich areas in human lymphoid tissues. Moreover, an intimate replication in human DCs/T-cell cocultures. First, iDCs were pre- contact between DCs and CD4 þ T cells has been shown to treated with the HIV-1 reverse transcriptase inhibitor efavirenz promote a very efﬁcient transmission of HIV-1 by locally con- (EFV) during the virus pulsing period. This treatment will abro- centrating virus, viral receptor and coreceptors, as well as certain gate de novo virus production in iDCs without altering transfer of adhesion molecules. viruses located on their surface or within their endosomal It is of interest to note that it has been shown that Milt reduces apparatus and the subsequent replication in CD4 þ T cells. This HIV-1 production in macrophages via downstream activation of treatment completely abolished virus production in iDCs cultured Akt kinase (Chugh et al., 2008). Since DCs are crucial actors in alone and a reduction in HIV-1 replication was also seen in HIV-1 mucosal transmission and the overall pathogenesis of the cocultured cells (data not shown). Interestingly, a comparable disease and given that CD4þ T cells act as a major cellular diminution in virus production was detected in both untreated reservoir for HIV-1, we analyzed the capacity of Mitl to affect and EFV-treated cocultures exposed to Milt (Fig. 2A). Therefore, it HIV-1 propagation in cocultures made of human immature can be proposed that Milt is not modulating susceptibility of iDCs monocyte-derived DCs and autologous CD4þ T cells. We report to virus infection and is thus most likely exerting its inhibitory here that Milt diminishes HIV-1 production in such cocultured effect in CD4 þ T cells. To provide more direct evidence on the cells. Furthermore, we provide evidence that the Milt-dependent mechanism of action of Milt, iDCs were initially exposed to fully reduction in virus replication is mainly affecting virus production infectious X4-tropic virions (i.e. NL4-3) and next cocultured with in CD4þ T cells and is at least partly linked with secretion of type- autologous CD4 þ T cells in presence of various concentrations of I interferon by DCs. Milt. The rationale for this experimental setup is based on the

Results

Milt reduces HIV-1 replication in cocultures of iDCs and CD4þ T cells. Milt is currently one of the first-line drug for the treatment of VL in HIV-1-infected and non-infected patients. In an attempt to define whether Milt can possibly affect the life cycle of HIV-1, we studied its effect on virus replication in the context of cocultures made of iDCs and autologous CD4 þ T cells. To this end, iDCs were initially pulsed with fully competent R5-tropic virions (i.e. NL4-3Balenv) and next exposed to increasing doses of Milt. Finally, immature monocyte-derived DCs (iDCs) were cocultured with autologous CD4 þ T cells. Results showed a statistically significant dose-dependent inhibition of HIV-1 replication in such cocultured cells (Fig. 1). It should be noted that Milt and B18R

Fig. 2. Milt modulates virus replication primarily in CD4þ T cells. (A) First, iDCs were either left untreated (Ctrl) or treated with the antiviral agent EFV. Second, cells were pulsed for 60 min with NL4-3Balenv and either left untreated (Ctrl) or treated with the listed concentrations of Milt. Finally, iDCs were cocultured with autologous CD4þ T cells. Virus production was estimated by measuring p24 levels Fig. 1. HIV-1 replication in human cocultured cells is reduced by Milt. First, iDCs in the culture supernatants at 4 day following initiation of the coculture. (B) iDCs were pulsed for 60 min with NL4-3Balenv and either left untreated (Ctrl) or were first pulsed for 60 min with NL4-3 and either left untreated (Ctrl) or treated treated with the listed concentrations of Milt. Second, cells were cocultured with with the listed concentrations of Milt. Next, cells were cocultured with autologous autologous CD4þ T cells and virus production was estimated by measuring p24 CD4þ T cells and virus production was estimated by measuring p24 levels in the levels in the culture supernatants at the indicated time points. Virus production at culture supernatants at the indicated time points. Virus production at day day 2 following initiation of the coculture is depicted in the small insert (upper left 2 following initiation of the coculture is depicted in the small inserts (upper left part). Data shown represent the means7SEM of triplicate samples and are part). Data shown represent the means7SEM of triplicate samples and are representative of six independent experiments. Asterisks denote statistically representative of five independent experiments. Asterisks denote statistically significant differences from the cells infected with HIV-1 only (*Po0.05; significant differences from the cells infected with HIV-1 only (*Po0.05; **Po0.01). **Po0.01). R. Garg, M.J. Tremblay / Virology 432 (2012) 271–276 273 previous report showing that iDCs are refractory to productive infection with X4-using variants (Pion et al., 2007). A dose- dependent reduction in HIV-1 replication was still seen in cocultures treated with Milt (Fig. 2B), which supports the idea that Milt is affecting HIV-1 replication in CD4þ T cells. To validate that HIV-1 production in CD4þ T cells is decreased upon a coculture step with Milt-treated iDCs, we performed another set of experiments where autologous CD4þ T cells were first pulsed with HIV-1 and then cocultured with iDCs either left untreated or treated with Milt. Moreover, this set of experiments was also aimed at assessing whether a cell-to-cell contact is needed to see the Milt-mediated diminution in virus production. To do so, we used permeable cell supports with a membrane pore size of 0.4 mm, which allows the crossing of virions and soluble factors but not that of cells (Ramirez-Pineda et al., 2004). Results depicted in Fig. 3 demonstrate that viral replication in CD4 þ T cells is similarly decreased by a coculture step with Milt-loaded iDCs when cells are in a close physical contact. When iDCs were physically separated from CD4 þ T cells by a permeable membrane, virus production was still diminished by a Milt treatment. Thus, these results corroborate that the inhibitory effect of Milt in cocultured cells is due to a repression of virus production in CD4 þ T cells and suggest that this process is caused by a Milt-mediated release of a soluble factor by iDCs. Milt-induced diminution of virus replication is partially mediated by type-I IFN. It has been reported that Milt drives secretion of IFN-g, TNF-a and IL-12 production from macrophages (Wadhone Fig. 4. Milt-induced inhibition of virus replication in cocultured cells is partially et al., 2009). Consequently, we hypothesized that the observed mediated by type-I IFN. (A) Cocultured cells were either left untreated or treated decrease in viral replication might be due to production by iDCs of with Milt for 6 h. Next, supernatants were collected and the levels of type-I IFN TM a soluble factor displaying an anti-HIV-1 activity in CD4þ T cells. were quantified through the use the HEK-Blue IFN-a/b cells. Data shown represent the means7SEM of triplicate samples and are representative of five Given that type-I IFN is recognized as a powerful inhibitor of independent experiments performed with different donors. (B) First, iDCs were in vitro and in vivo HIV-1 infection, our next series of investiga- pulsed for 60 min with NL4-3Balenv and either left unexposed or exposed to Milt. tions was aimed at assessing the capacity of Milt to induce Next, iDCs were cocultured with autologous CD4þ T cells and either left untreated secretion of type-I IFN in iDCs with the use of the HEK-BlueTM or treated with B18R. Virus production was estimated by measuring p24 levels in the culture supernatants at 4 day following initiation of the coculture. The results IFN- /b indicator cell line. We observed that Milt acts indeed as a a shown represent the means7SEM of triplicate samples and are representative of good inducer of type-I IFN following treatment of human iDCs four separate experiments performed with different donors and are expressed as (Fig. 4A). To validate the putative role of type-I IFN in the relative viral production compared to cocultured cells exposed to HIV-1 but left observed Milt-dependent reduction of HIV-1 production in cocul- untreated with Milt and B18R. Asterisks denote statistically significant data tured cells, virus transfer experiments were performed with the (*Po0.05; **Po0.01). soluble vaccinia virus-encoded protein B18R that has been demonstrated to inhibit the antiviral activity and cellular binding prior to initiation of the coculture step with autologous CD4 þ T of the type-I IFN of different mammalian species (e.g. IFN-a, IFN-b cells. Data from Fig. 4B indicate that the Milt-mediated reduction and IFN-o)(Alcami et al., 2000; Symons et al., 1995). B18R was in de novo virus production seen in cocultured cells was asso- added simultaneously with Milt in iDCs inoculated with HIV-1 ciated with secretion of type-I IFN. A higher concentration of B18R did not further increase virus production (data not shown), thus suggesting that the Milt-directed inhibition of virus production in cocultured cells is not only due to production of type-I IFN by iDCS but also to another unknown soluble factor.

Discussion

Milt is an alkylphosphocholine analog that was originally developed as an antitumor agent but proved to be clinically ineffective against tumors, with intolerable adverse effects. Data from animal and in vitro experimental studies suggest that Milt displays also broad anti-fungal (e.g. Cryptococcus, Candida, Asper- gillus and Fusarium) and anti-protozoal properties (e.g. Plasmo- dium, Trypanosoma and Leishmania)(Blaha et al., 2006; Saraiva Fig. 3. Milt-dependent decrease in virus production seen in cocultured cells is due to a soluble factor. First, CD4þ T cells were incubated for 2 h with NL4-3. Next, et al., 2002; Widmer et al., 2006). Milt has been used in multiple CD4þ T cells were cocultured with iDCs in the presence of the listed concentration clinical trials and is now approved for treatment of leishmaniasis of Milt for 4 day either in cell-to-cell contact or non-contact conditions. Virus in Germany, India and Columbia. Leishmaniasis is now recognized production was estimated by measuring p24 levels in the culture supernatants at as one of the leading HIV-1-associated opportunistic disease the indicated time points. Data shown represent the means7SEM of triplicate in various countries. It is known that HIV-1-infected patients samples and are representative of five independent experiments. Asterisks denote statistically significant differences from the cells infected with HIV-1 only who are receiving combined therapy have fewer opportunistic (**Po0.01). infections. For example, it has been reported that the HIV-1 274 R. Garg, M.J. Tremblay / Virology 432 (2012) 271–276 protease inhibitors significantly inhibit the intracellular survival type-I IFN is involved in the inhibitory effect mediated by this of Leishmania parasites (Trudel et al., 2008; Valdivieso et al., 2010; drug. Altogether our data suggest a partial role of type-I IFN in the White et al., 2011). Milt tolerability and efficacy in immunocom- Milt-mediated inhibition of HIV-1 replication seen in a coculture petent individuals have already been proven in earlier studies (Jha system consisting of iDCs and autologous CD4 þ T cells. et al., 1999; Le Fichoux et al., 1998; Murray and Delph-Etienne, It was shown previously that patients dually infected with 2000) but there is still many unanswered questions in immuno- HIV-1 and Leishmania, who had experienced failure of standard compromised persons. These reports together with the idea that leishmaniasis treatment, displayed an initial response rate of 64% the efficacy of drugs aimed at controlling each pathogen remains and a cure rate of 43% during the first treatment cycle with Milt largely undefined in dually infected persons underlined the need (Sindermann et al., 2004). Milt is now recognized as the first oral for further investigation. agent that appears to be highly effective and well tolerated for the DCs are central for the development of pathogen-specific treatment of co-infected patients. The current work demonstrates immune responses and are well equipped for activation of both for the first time that Milt can, in addition to its already known the innate and adaptive immune response (Mellman and anti-leishmanicidal properties, restrict also HIV-1 propagation Steinman, 2001). It has been demonstrated that human pathogens and shed light on the mechanisms by which this compound can targeting DCs, such as HIV-1, have evolved strategies to impair DC modulate virus replication. These results add new and interesting functions, thereby enhancing their capacity to persist and evade in vitro insights into the possible inhibitory activity of Milt immune surveillance. DCs have been reported as playing an against HIV-1 and suggest studying its efficacy on others cell important role in the pathogenesis of AIDS, favoring both the targets (e.g. macrophages) to validate its use on a larger scale in initial establishment and spread of the infection and the devel- individuals co-infected with HIV-1 and Leishmania. However, the opment of antiviral immunity. In the present study, we investi- precise clinical relevance of our present findings remains to be gated whether Milt treatment can modulate HIV-1 replication determined. when iDCS are cocultured with autologous CD4 þ T cells. We provide the first evidence showing that Milt represses HIV- 1 replication in iDCs-T-cell cocultures of human origin. To define Material and methods the precise contribution of de novo virus production from iDCs in the Milt-dependent down-regulatory effect on HIV-1 replication, Reagents. Milt was obtained from Alexis Biochemicals (San coculture experiments were performed in presence of the anti- Diego, CA) while B18R was purchased from eBiosciences (San retroviral compound EFV. Results with EFV-treated cells and Diego, CA). Recombinant human interleukin-2 (rhIL-2), EFV and experiments performed with X4-tropic virions led us to conclude azidothymidine (AZT) were obtained from the NIH AIDS Reposi- that the Milt-directed effect in virus production is independent of tory Reagent Program (Germantown, MD). Interferon-gamma productive infection of iDCs. Interestingly, our data indicate also (IFN-g) and IL-4 were both purchased from R&D systems (Min- that a close physical contact between Milt-treated iDCs and CD4 þ neapolis, MN), whereas granulocyte macrophage-colony stimu- T cells is not necessary to diminish HIV-1 production, therefore lating factor (GM-CSF) was a generous gift from Cangene indicating that the observed reduction in virus replication is (Winnipeg, MB). Lipopolysaccharide (LPS) and phytohemaggluti- achieved through the secretion of a soluble factor. This postulate nin-L (PHA-L) were obtained from Sigma (St-Louis, MO). The is supported by the previous observation that Milt induces culture medium consisted of RPMI-1640 supplemented with production of numerous cytokines such as TNF-a, GM-CSF, IL-12 10% fetal bovine serum (FBS), penicillin G (100 U/mL), strepto- and IFN-g (Beckers et al., 1994; Hochhuth et al., 1992; Wadhone mycin (100 U/mL) and glutamine (2 mM), which were all pur- et al., 2009; Zeisig et al., 1995). The human IFN network is known chased from Wisent (St-Bruno, QC), and primocine (Amaxa as one of the fastest responding branch of our immune defense to Biosystems, Gaithersburg, MD). control an invading pathogen. The IFN system is an extremely Cells. Human embryonic kidney 293 T cells were cultured in powerful antiviral response that is capable of controlling most, if Dulbecco’s modified Eagle’s medium (DMEM) supplemented with not all, virus infections in the absence of adaptive immunity. 10% FBS (Wisent, St-Bruno, QC). DCs were generated from mono- Type-I IFN (e.g. a and b) are produced by many cell types, while cytes obtained from the blood of healthy donors. Briefly, CD14þ type-II IFN (i.e. g) is more restricted since it is produced by cells (i.e. monocytes) were isolated from peripheral blood mono- immune cells. Type-I IFN comprises a large group of molecules nuclear cells, using a monocyte-positive selection kit according to that includes IFN-a and -b as well as IFN-o,-E,-t,-d and -k. The the manufacturer’s instructions (CD14-positive selection kit; IFN-a and -b genes are induced directly in response to viral StemCell Technologies Inc., Vancouver, BC). Purified CD14 þ cells infection, whereas IFN-o,-E,-d and -k play less well-defined were cultured in RPMI-1640 medium supplemented with 10% roles, such as regulators of maternal recognition in pregnancy. We FBS, GM-CSF (1000 U/ml) and IL-4 (200 U/ml) for 7 day to obtain show here using HEK-BlueTM IFN a/b cells that the Milt-mediated iDCs as previously described (Gilbert et al., 2007). Autologous restriction of HIV-1 replication is due in part to an endogenous CD4þ T cells were isolated with a negative selection kit (Stem Cell production of type-I IFN. Indeed, this indicator cell line has Technologies) and activated with the mitogenic agent PHA-L (1 allowed us to determine that treatment of iDCs with Milt induces mg/ml) and IL-2 (30 U/ml) for 48 h prior to their use. HEK-BlueTM a rapid production of type-I IFN (i.e. as early as 3 h following IFN-a/b cells (InvivoGen, San Diego, CA) were maintained in exposure to the drug) reaching a peak at 6 h (data not shown). Dulbecco’s modified Eagle medium (Invitrogen) supplemented Previous studies revealed that treatment of human cells with with 10% FBS, glutamine (2 mM), penicillin G (100 U/ml) and type-I IFN restricts HIV-1 replication via blocking both early and streptomycin (100 mg/ml). Culture media used for HEK-BlueTM late stages of the virus replicative cycle (Agy et al., 1995; Baca- IFN-a/b cells was supplemented with 30 mg/ml of blasticidin and Regen et al., 1994; Coccia et al., 1994; Shirazi and Pitha, 1992). 100 mg/ml of Zeocin. Treatment of iDCs with the type-I IFN-binding receptor B18R Viral preparations. Virus stocks were produced upon transient confirms the importance of such a soluble factor in the Milt- calcium phosphate transfection of 293 T cells with full-length dependent inhibitory effect on HIV-1 gene expression. 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