Anti-HIV Activity of Cucurbitacin-D Against Cigarette Smoke Condensate-Induced HIV Replication in the U1 Macrophages

viruses

Article Anti-HIV Activity of Cucurbitacin-D against Cigarette Smoke Condensate-Induced HIV Replication in the U1 Macrophages

Sunitha Kodidela *,† , Namita Sinha †, Asit Kumar and Santosh Kumar *

The Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA; [email protected] (N.S.); [email protected] (A.K.) * Correspondence: [email protected] (S.K.); [email protected] (S.K.) † These authors contributed equally to this work.

Abstract: Chemodietary agents are emerging as promising adjuvant therapies in treating various disease conditions. However, there are no adjuvant therapies available to minimize the neurotoxicity of currently existing antiretroviral drugs (ARVs). In this study, we investigated the anti-HIV effect of a chemodietary agent, Cucurbitacin-D (Cur-D), in HIV-infected macrophages using an in-vitro blood–brain barrier (BBB) model. Since tobacco smoking is prevalent in the HIV population, and it exacerbates HIV replication, we also tested the effect of Cur-D against cigarette smoke condensate (CSC)-induced HIV replication. Our results showed that Cur-D treatment reduces the viral load in a dose-dependent (0–1 µM) manner without causing significant toxicity at <1 µM concentration. Further, a daily dose of Cur-D (0.1 µM) not only reduced p24 in control conditions, but also reduced µ µ CSC (10 g/mL)-induced p24 in U1 cells. Similarly, Cur-D (single dose of 0.4 M) significantly reduced the CSC (single dose of 40 µg/mL)-induced HIV replication across the BBB model. In β Citation: Kodidela, S.; Sinha, N.; addition, treatment with Cur-D reduced the level of pro-inflammatory cytokine IL-1 . Therefore, Kumar, A.; Kumar, S. Anti-HIV Cur-D, as an adjuvant therapy, may be used not only to suppress HIV in the brain, but also to reduce Activity of Cucurbitacin-D against the CNS toxicity of currently existing ARVs. Cigarette Smoke Condensate-Induced HIV Replication in the U1 Keywords: Cucurbitacin-D; HIV; blood–brain barrier model; cytokines/chemokines; p24; macrophages; Macrophages. Viruses 2021, 13, 1004. cigarette smoke condensate https://doi.org/10.3390/v13061004

Academic Editors: Maria Cecilia Garibaldi Marcondes and 1. Introduction Marcus Kaul The prevalence of HIV-associated neurocognitive disorders (HAND) is increasing despite the successful implementation of antiretroviral therapy (ART) [1,2]. There is an Received: 23 February 2021 evidence of transmigration of CD14+CD16+ monocytes in the central nervous system (CNS), Accepted: 25 May 2021 Published: 27 May 2021 which perpetuates the neuropathogenesis of HIV [3]. However, there is a lack of association between neurocognitive impairment and virological and immunological indicators, which

Publisher’s Note: MDPI stays neutral suggests the progression of neuronal damage in HIV-positive subjects regardless of the with regard to jurisdictional claims in success of ART [4]. Furthermore, the detection of viral RNA is observed in the brains published maps and institutional affil- of subjects with complete suppression of plasma viral load by ART [5]. The increased iations. viral load in the brain is mainly due to the inability of the current ART drugs to cross the blood–brain barrier (BBB) and suppress the virus efficiently [6,7]. The presence of even very low levels of viral replication in the CNS could result in neural injury or dysfunction due to prolonged exposure to inflammatory responses and neurotoxic viral proteins. Cigarette smoking is prevalent among HIV-positive subjects [8] and it has been re- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. ported to increase HIV replication and its associated conditions by various mechanisms, This article is an open access article including oxidative stress [9–11]. Furthermore, smokers with HIV are more likely to have distributed under the terms and a poorer response to ART and subsequently to develop more serious comorbidities and conditions of the Creative Commons premature death than non-smokers with HIV [12,13]. Moreover, smoking was associ- Attribution (CC BY) license (https:// ated with a higher risk of progression from asymptomatic neurocognitive impairment to creativecommons.org/licenses/by/ symptomatic HAND in people living with HIV [14]. Although the cessation of cigarette 4.0/). smoking is the ultimate cure for smoking-exacerbated HIV complications [12], it is difficult

Viruses 2021, 13, 1004. https://doi.org/10.3390/v13061004 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 1004 2 of 14

to achieve. Furthermore, the current use of ART drugs is ineffective against the virus in the brain, and it can induce neurotoxicity [15]. Recently, FDA has approved the first long-acting drug combo for HIV, monthly shots which are beneficial for people who are more likely to have a reduced compliance to the treatment, including people who smoke cigarette [16]. However, relatively high costs of the monthly shots may limit their use among the HIV population. Besides, this drug formulation does not solve the problem of ineffective treatment of HIV in the brain. Thus, there is a need to constantly develop new drugs or drug-like compounds that can, alone or in the presence of current ART drugs, effectively suppress HIV, as well as smoking-exacerbated HIV replication and its associated complications, including HAND. Naturally occurring dietary compounds have gained increasing attention for the prevention of various types of cancers and viral infections, including HIV [17–20]. Cucurbitacin- D (Cur-D) is a tetracyclic triterpene commonly found in the cucurbitaceae family, which has been used in conventional medicine for decades [17,21]. However, no study has demon- strated its anti-HIV activity. In this study, we show, for the first time, the potential anti-HIV activity of Cur-D across the in-vitro BBB model in the absence and presence of tobacco constituents.

2. Materials and Methods 2.1. Chemicals and Reagents The cigarette smoke condensate (CSC) was purchased from Murty Pharmaceuticals (Lexington, KY, USA). The Cur-D was obtained from Sigma-Aldrich (St. Louis, MO, USA). The HIV Type 1 p24 Antigen ELISA kit to assess HIV viral load was purchased from ZeptoMetrix Corporation (Buffalo, NY, USA). Pierce Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit was purchased from ThermoFisher Scientific (Grand Island, NY, USA). Cell culture reagents including the Roswell Park Memorial Institute (RPMI) 1640 media were purchased from Corning Inc. (Tewksbury, MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from American Type Culture Collection. Fetal bovine serum (heat-inactivated) was bought from Atlanta Biologicals (Atlanta, GA, USA). L-glutamine and penicillin-streptomycin (P/S) were purchased from Fisher Scientific.

2.2. Cell Culture and Treatment U1 cells, which are U937 cells chronically infected with the human immunodeﬁ- ciency virus type 1 (HIV-1), were procured from the NIH AIDS Reagent Program (Ger- mantown, MD, USA). The U1 cell lines have been widely used by our group and many other research groups to study the role of drug abuse, including tobacco smoking in HIV replication [10,11,22–25]. Furthermore, the results obtained using these cells have been successfully replicated in HIV-infected primary macrophages [10,11,24]. The U1 cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 1% L- glutamine, and 2% penicillin/streptomycin. To differentiate the cells into macrophages, 0.3 million cells were seeded in 0.4 mL of media containing 100 nM phorbol 12-myristate 13-acetate) in each well of a 12-well plate. After 3 days of differentiation, the media was removed, cells were washed with phosphate buffer saline, and the fresh media was added to the differentiated cells. After adding the fresh media to the cells, the cells were incubated for 3–4 h before starting the treatment. The differentiated cells were treated with control (DMSO), CSC (10 µg/mL), Cur-D (0.1 µM), and CSC (10 µg/mL) + Cur-D (0.1 µM) every day for 3 days. In a separate experiment, we also used established ART drug regimen darunavir-ritonavir (DRV-RTV) (12 µg/mL and 4 µg/mL, respectively) as positive control and compared the data with Cur-D. We used DRV as a positive control because the literature suggests that DRV was detectable in cerebrospinal ﬂuid samples of HIV subjects [26]. The p24 antigen levels were determined in the collected culture supernatant using the p24 ELISA kit. We procured mouse and human brain endothelial cells (CRL-2299) and astrocytes (CRL-2541) from ATCC (Manassas, VA, USA). These cells are validated by the ATCC and Viruses 2021, 13, x 3 of 15

subjects [26]. The p24 antigen levels were determined in the collected culture supernatant Viruses 2021, 13, 1004 using the p24 ELISA kit. 3 of 14 We procured mouse and human brain endothelial cells (CRL-2299) and astrocytes (CRL-2541) from ATCC (Manassas, VA, USA). These cells are validated by the ATCC and have been widely used to prepare the in vitro BBB model, including by our group [25]. have been widely used to prepare the in vitro BBB model, including by our group [25]. These cells were cultured in DMEM media supplemented with 10% FBS and 1% PS solu- These cells were cultured in DMEM media supplemented with 10% FBS and 1% PS solution tion and incubated at◦ 37 °C with 5% CO2 before building in vitro BBB model. and incubated at 37 C with 5% CO2 before building in vitro BBB model.

InIn Vitro Vitro BBB Model WeWe usedused TranswellTranswell®-COL®-COL collagen-coated collagen-coated 0.4 0.4µm µm pore pore polytetrafluoroethylene polytetrafluoroethylene mem- membranebrane insert insert (Sigma-Aldrich) (Sigma-Aldrich) to prepare to prepare an in vitro an inBBB vitro model BBB as model described as described previously previ- [27]. ouslyMouse [27]. model: First, we used mouse endothelial and astrocytic-cells to represent our futureMouse proposed model: work First, with we used the HIV mouse mice endothe modellial to and study astrocytic-cells the pharmacokinetics, to represent tissue our futuredistribution, proposed and work efficacy with of the Cur-D HIV on mice viral mo suppression.del to study Briefly,the pharmacokinetics, the mouse astrocytes tissue distribution,(2 × 105 cells/well) and efficacywere of seeded Cur-D on on the viral bottom suppression. of 12-well Briefly, plates. the mouse After 24 astrocytes h of adhe- (2 ×sion, 105 cells/well) mouse endothelial were seeded cells on (2 the× 10bottom5 cells/well of 12-well) were plates. seeded After onto 24 h the of upperadhesion, side mouse of the endothelialTranswell® -cells COL (2 inserts, × 105 cells/well) and the inserts were wereseeded placed onto inthe a 12-wellupper side plate of containing the Transwell astro-®- COLcytes. inserts, These cellsand the constitute inserts thewere BBB placed model in anda 12-well were grownplate containing for 5 days astrocytes. to achieve These ~90% cellsconfluency. constitute After the achieving BBB model 90% and confluency, were grown the upper for 5 insertsdays to containing achieve ~90% endothelial confluency. cells Afterwere achieving transferred 90% to theconfluency, wells containing the upper U1-differentiated inserts containing macrophages. endothelial cells Transendothe- were trans- ferredlial electrical to the wells resistance containing (TEER) U1-differentiat using EVOM2ed Epithelial macrophages. Voltohmmeter Transendothelial (World Precisionelectrical resistanceInstruments, (TEER) Sarasota, using FL) EVOM2 was measuredEpithelial asVoltohmmeter described [27 (World]. A mean Precision TEER Instruments, value of 100 Sarasota,to 120 Ohms FL) ×wascm measured2 was observed as described in the confluent[27]. A mean BBB TEER model value and publishedof 100 to 120 in Ohms our pre- × cmvious2 was reports observed [27]). in To the determine confluent theBBB efficacy model and of Cur-D published on CSC-induced in our previous viral reports replication, [27]). Toendothelial determine cells the inefficacy the upper of Cur-D inserts on wereCSC-in exposedduced viral to a singlereplication, dose ofendothelial control (DMSO), cells in theCSC upper (40 µ insertsg/mL), were Cur-D exposed (0.4 µM), to a and single CSC dose (40 ofµ g/mL)control +(DMSO), Cur-D (0.4 CSCµ M)(40 andµg/mL), observed Cur- Dfor (0.4 3 days. µM), Inand this CSC case, (40 we µg/mL) used a+ higherCur-D dose(0.4 µM) of CSC, and becauseobserved a lowerfor 3 days. CSC In dose this shows case, weinability used a to higher cross thedose BBB of CSC, and effectivelybecause a lower suppress CSC HIV dose across shows the inability BBB. HIV-1 to cross viral the loads BBB andwere effectively measured suppress every day HIV in the across cell culturethe BBB. supernatant HIV-1 viral from loads the were bottom measured chamber every using day a inp24 the ELISA cell culture kit. supernatant from the bottom chamber using a p24 ELISA kit. HumaHuma model: model: After After establishing establishing the the effect effect of of Cur-D Cur-D against against CSC-induced CSC-induced HIV HIV repli- repli- cationcation in in the the mouse BBB model, we develop developeded a a human BBB model (Figure 11)) toto mimicmimic thethe human human system. system. In In this this model, model, we we tested tested th thee anti-HIV anti-HIV effect effect of of Cur-D Cur-D and compared it with established ART ART regimen (DRV-RTV) as a positive control. For this, we used human astrocytesastrocytes and and endothelial endothelial ce cellslls and and followed followed the the same same procedure procedure mentioned mentioned above above to de- to velopdevelop a human a human BBB BBB model. model. We We treated treated the the endo endothelialthelial cells cells in in the the upper upper inserts inserts with with a singlesingle dose of controlcontrol (DMSO),(DMSO), CSC CSC (40 (40µ µg/mL),g/mL), Cur-D Cur-D (0.4 (0.4µ µM),M), CSC CSC (40 (40µ g/mL)µg/mL) + + Cur-D Cur- µ µ µ D(0.4 (0.4M), µM), and and DRV-RTV DRV-RTV (12 (12 g/mLµg/mL and and 4 4 µgg/mL,/mL, respectively)respectively) and observed for 2 2 days. days. The human BBB model was relatively sensitive than the mouse BBB model, and thus we couldcould only only continue continue the treatment for up to 2 days.

FigureFigure 1. 1. GraphicalGraphical representation representation of th thee in vitro human BBB model. 2.3. HIV Type 1 p24 ELISA 2.3. HIV Type 1 p24 ELISA The HIV-1 p24 antigen level in the supernatant collected from U1 cells was measured The HIV-1 p24 antigen level in the supernatant collected from U1 cells was measured using an HIV-1 p24 Antigen ELISA kit (Zeptometrix Corporation, Buffalo, NY, USA). The using an HIV-1 p24 Antigen ELISA kit (Zeptometrix Corporation, Buffalo, NY, USA). The kit consists of microwells that are coated with a monoclonal antibody speciﬁc for HIV-1 kit consists of microwells that are coated with a monoclonal antibody specific for HIV-1 P24 antigen. The samples containing the HIV-1 p24 antigen were added to these wells. The captured viral antigen was sequentially incubated with biotin-labeled human antibody to HIV-1 for 1 h, streptavidin conjugated to horseradish peroxidase for 30 min at 37 ◦C, and tetramethylbenzidine substrate for 30 min in the dark. The optical density of every well was measured at 450 nm and compared against the standard curve to determine the p24 Viruses 2021, 13, 1004 4 of 14

(pg/mL) levels in the samples. The viral load was expressed as a percentage of HIV-1 P24 levels observed in DMSO-treated control wells.

2.4. LDH Cytotoxicity Assay Cytotoxicity was measured in the cell culture supernatant collected from differentiated U1 macrophage media using the Pierce Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (ThermoFisher Scientiﬁc, Grand Island, NY, USA) following the manufacturer’s protocol. Brieﬂy, 50 µL of the collected culture supernatant was mixed with 50 µL of the LDH reaction mixture in a 96-well plate and incubated at room temperature for 30 min. Then, the 50 µL of LDH stop solution were added to stop the reaction. The absorbance was measured at 490 nm and 680 nm using a microplate reader (Cytation™ 5 Cell Imaging Multi-Mode Reader, BioTek, VT, USA). Higher absorbance suggests higher toxicity.

2.5. Western Blotting To determine the expression of IL-1β, catalase, and SOD1, an equal amount of protein (10 µg) was used from control (DMSO), CSC, and Cur-D treated differentiated U1 macrophages. The proteins from different study groups were loaded onto polyacrylamide gel (4% stacking, 10% resolving gel), run for 90 min at 150 V, and then transferred to polyvinyl ﬂuoride membrane using a current of 0.35 Amp for 90 min. After the proteins were transferred to the membrane, it was incubated with 5–10 mL of Li-Cor blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h to avoid the nonspeciﬁc binding of antibodies to its surface. The membrane was then incubated overnight at 4 ◦C with target primary antibodies (IL-1β Rabbit Pab, 1:500 dilution, proteintech, catalog#16806-1-AP; SOD1 mouse Mab, 1:200 dilution, Santa Cruz Biotechnology, catalog #sc-101523; Catalase mouse Mab, 1:100 dilution, Santa Cruz Biotechnology, catalog # sc-365738; β-Actin Mouse mAb.1:2000 dilution, Cell Signaling, Catalog #3700) at 4 ◦C overnight. The next day, the blots were washed with PBS containing 0.2% Tween-20 (PBST) three times and then incubated with the corresponding secondary antibodies (Goat anti-Mouse Mab, Goat anti-Rabbit Mab, 1:10,000 dilution, LI-COR Biosciences) for 1 h at room temperature in the dark. The membrane was washed again with PBST and the blots were scanned using Image Studio Lite version 4.0 in a Li-Cor Scanner (LI-COR Biosciences). The densitometric data was obtained from the Image Studio Lite software. Actin was used as an internal loading control to normalize the expression of IL-1β, catalase, and SOD1 proteins.

2.6. Cytokine Analysis The protein levels of various cytokines and chemokines such as pro-inflammatory: TNF-α, IL-1β, IL-8, IL-6; anti-inflammatory: IL-1ra, IL-10; and chemokines: MCP-1, and RANTES were measured from the culture media (25 µL) of differentiated U1 macrophages using Human Custom Procartaplex 8-plex (Invitrogen, ThermoFisher Scientific, Grand Island, NY, USA). Following the manufacturer’s protocol, samples, standards, and magnetic beads were added to the 96-well ELISA plate and mixed well on a plate shaker for 1 h at room temperature, followed by overnight incubation at 4 ◦C. The next day, the beads were washed, followed by the addition of the detection antibody, streptavidin-PE, and reading buffer, with subsequent washing off of reagents at each step. The concentration (pg/mL) of the cytokines and chemokines were measured using a Magpix system, and the data were analyzed using the xPONENT® software.

2.7. Statistical Analysis The GraphPad Prism 5 (GraphPad Software; La Jolla, CA, USA) was used to perform all statistical analyses and to plot graphs. The data are presented as mean ± SEM. One-way ANOVA with Tukey’s post-hoc test was applied to compare between multiple groups; p < 0.05 is considered signiﬁcant. Viruses 2021, 13, x 5 of 15

Viruses 2021, 13, x 5 of 15

2.7. Statistical Analysis 2.7. StatisticalThe GraphPad Analysis Prism 5 (GraphPad Software; La Jolla, CA, USA) was used to perform all statisticalThe GraphPad analyses Prism and 5to (GraphPad plot graphs. Software; The data La are Jolla, presented CA, USA) as wasmean used ± SEM. to perform One- wayall statistical ANOVA analyses with Tukey’s and to post-hocplot graphs. test Thewas data applied are presentedto compare as meanbetween ± SEM. multiple One- Viruses 2021, 13, 1004 groups;way ANOVA p < 0.05 withis considered Tukey’s significant.post-hoc test was applied to compare between multiple5 of 14 groups; p < 0.05 is considered significant. 3. Results 3.1.3.3. ResultsResults Cur-D Does Not Exhibit Cytotoxicity in U1 Macrophages 3.1.3.1. Cur-DCur-DSince there DoesDoes NotNotis a ExhibitExhibitlack of Cytotoxicity Cytotoxicitydata on the in insafe U1U1 MacrophagesdoseMacrophages of Cur-D in U1 macrophages, we per- formedSinceSince an LDH therethere assay isis aa lacklack to analyze ofof datadata the onon cytotoxicity thethe safesafe dosedose ofof Cur-Dof Cur-DCur-D on in inU1 U1U1 cells. macrophages,macrophages, For this, we we wetreated per-per- U1formedformed cells an withan LDH LDH different assay assay to concentrationsto analyze analyze the the cytotoxicity cytotoxicity of Cur-D (0, of of 0.01, Cur-D Cur-D 0.05, on on 0.1, U1 U1 0.5, cells. cells. and For For 1 this, µM)this, we everywe treated treated day forU1U1 3 cellscells days. withwith We differentdifferent observed concentrationsconcentrations that treatment of of of Cur-D Cur- U1 cellsD (0, (0, with 0.01, 0.01, 0.05,0.01 0.05,– 0.1,1 0.1, µM 0.5, 0.5, of andCur-Dand 11µ µM) M)for 1, everyevery 2, and dayday 3 daysforfor 33 days.diddays. not WeWe show observed observed a statistically that that treatment treatment significant of of U1 U1 increase cells cells with with in LDH 0.01–1 0.01 activity–1µ µMM of of (Figure Cur-D Cur-D 2), for for suggesting 1, 1, 2, 2, and and 3 3 nodaysdays detectable diddid notnot showshow cytotoxicity aa statisticallystatistically with signiﬁcantthesignificant selected increasein docreaseses. in Therein LDH LDH appearsactivity activity (Figureto (Figure be an2 ),2), inconsistent suggesting suggesting patternnono detectabledetectable of toxicity cytotoxicitycytotoxicity on day with1,with perhaps thethe selectedselected due to doses. dotheses. initial ThereThere stress appearsappears caused toto bebeby an antheinconsistent inconsistent treatment, patternwhichpattern is of ofa toxicitycommon toxicity on observationon day day 1, perhaps1, perhaps with due treatment due to the to initialthe with initial stressany xenobioticstress caused caused by agent. the by treatment, the treatment, which iswhich a common is a common observation observation with treatment with treatment with any with xenobiotic any xenobiotic agent. agent.

Figure 2. Effect of different doses of cucurbitacin-D (Cur-D) on cytotoxicity of U1 macrophages: U1-monocytesFigureFigure 2.2.Effect Effect were ofof differentdifferent plated dosesin doses 12-well of of cucurbitacin-D cucurbitacin-D plates (0.3 million (Cur-D) (Cur-D) cells/well) on on cytotoxicity cytotoxicity and differentiated of of U1 U1 macrophages: macrophages: to macro- U1- phages.monocytesU1-monocytes Differentiated were were plated plated U1 in macrophages 12-well in 12-well plates plates were (0.3 million (0.3treate milliond cells/well)with cells/well) different and anddoses differentiated differentiated of Cur-D to ranging macrophages. to macro- from 0.01Differentiatedphages.–1 µM Differentiated daily U1 for macrophages 3 days. U1 macrophagesThe wereLactate treated Dehydrog were with treate differentenased with (LDH) dosesdifferent release of Cur-D doses in the rangingof supernatantCur-D from ranging 0.01–1 was from µM measureddaily0.01– for1 µM 3 every days. daily day Thefor 3by Lactate days. the LDH The Dehydrogenase Lactate assay. Dehydrog (LDH)enase release (LDH) in the release supernatant in the supernatant was measured was every measured every day by the LDH assay. day by the LDH assay. 3.2. Treatment with Cur-D Reduces p24 Levels in U1 Cells 3.2. Treatment with Cur-D Reduces p24 Levels in U1 Cells 3.2. TreatmentTo determine with the Cur-D anti-HIV Reduces activity p24 Levels of Cu inr-D, U1 weCells treated U1 macrophages with 0.01– 1 µMTo Toof determine Cur-Ddetermine every the the day anti-HIV anti-HIV for 3 activitydays. activity We of Cur-D,observedof Cur-D, we a treatedwe dose-dependent treated U1 macrophages U1 macrophages reduction with in0.01–1 with the viral0.01µM– loadof1 µM Cur-D with of Cur-Devery Cur-D dayevery treatment for day 3 days. for in 3 1 Wedays. and observed 2We days observed (Figure a dose-dependent a 3).dose-dependent Treatment reduction with reduction 0.1, in 0.5, the inand viral the 1 load viralµM withforload 2 and Cur-Dwith 3 Cur-Ddays treatment showed treatment in a 1 significant and in 1 2 and days 2reduction days (Figure (Figure3 in). Treatmentthe 3). viral Treatment load. with There 0.1,with 0.5,was 0.1, and no0.5, significant 1andµM 1 forµM difference2for and 2 and 3 days 3in days the showed reductionshowed a signiﬁcant a ofsignificant viral load reduction reduction between in in the0.1 the vs. viral viral 0.5 load. µM load. and There There 0.1 wasvs. was 1 no µMno signiﬁcant significant of Cur-D indifferencedifference 3 days. Therefore, in in thethe reductionreduction we selected of of viral viral 0.1 load load µM between betwof cur-Deen 0.1 0.1for vs. vs.the 0.5 0.5subsequentµ µMM and and 0.1 0.1experiments. vs. vs. 11µ µMM of of Cur-D Cur-D inin 33 days.days. Therefore,Therefore, wewe selected selected 0.1 0.1µ µMM of of cur-D cur-D for for the the subsequent subsequent experiments. experiments.

Figure 3. Dose-dependent effect of Cur-D on HIV p24 le levels:vels: U1-monocytes were plated in 12-well platesFigure (0.3 3. Dose-dependentmillion cells/well) cells/well) effect and and ofdifferentiated differentiated Cur-D on HIV to to macrophages. p24 macrophages. levels: U1-monocytes Differentiated Differentiated were U1 U1 platedmacrophages macrophages in 12-well wereplates treated (0.3 million with with different differentcells/well) doses doses and of differentiated of Cur-D Cur-D ranging ranging to macrophages.from from 0.01 0.01–1–1 µM Differentiatedµ forM for3 days. 3 days. The U1 p24macrophages The levels p24 levels werewere measured treated with every every different day day by by doses the the p24 p24of Cur-DElisa Elisa kit. ranging kit. Vi Viralral loadfrom load was 0.01 was expressed–1 expressedµM for as3 days. asa percentage a percentageThe p24 oflevels viral of viral loadwere observed measured in everyDMSO-treated day by the control p24 Elisa wells. kit. The TheViral data data load shown shown was expressed represent represent asthe the a mean meanpercentage ±± SEMSEM of of ofviral three three load observed in DMSO-treated control wells. The data shown represent the mean ± SEM of three independent experiments. One-way ANOVA with Tukey’s post-hoc test was applied to compare between multiple groups. *, **, and *** represent p < 0.05, p ≤ 0.01, and p ≤ 0.001, respectively, when

compared to 0 µM of Cur-D.

3.3. Relative Anti-HIV Effect of Cur-D Compared to DRV/RTV Positive Control After establishing the anti-HIV effect of Cur-D at different doses and time points, we tested whether the anti-HIV activity of Cur-D is comparable to that of established ART Viruses 2021, 13, x 6 of 15 Viruses 2021, 13, x 6 of 15

independentindependent experiments. experiments. One-way One-way ANOVA ANOVA with with Tu Tukey’skey’s post-hoc post-hoc test test was was applied applied to to compare compare betweenbetween multiple multiple groups. groups. *, *, **, **, and and *** *** represent represent p p< <0.05, 0.05, p p≤ ≤0.01, 0.01, and and p p≤ ≤0.001, 0.001, respectively, respectively, when when comparedcompared to to 0 0µM µM of of Cur-D. Cur-D.

Viruses 2021, 13, 1004 3.3.3.3. Relative Relative Anti-HIV Anti-HIV Effect Effect of of Cur- Cur-DD Compared Compared to to DRV/RTV DRV/RTV Positive Positive Control Control 6 of 14 AfterAfter establishing establishing the the anti-HIV anti-HIV effect effect of of Cu Cur-Dr-D at at different different doses doses and and time time points, points, we we testedtested whether whether the the anti-HIV anti-HIV activity activity of of Cur-D Cur-D is is comparable comparable to to that that of of established established ART ART drugs, with DRV-RTV as a positive control. To test this, we treated U1 macrophages with drugs,drugs, with with DRV-RTV DRV-RTV as as a a positive positive control. control. To To test test this, this, we we treated treated U1 U1 macrophages macrophages with with 0.1 µM of Cur-D and 3 µg/mL of DRV (Cmax of oral 400 mg QD), 1 µg/mL of RTV (Cmax 0.10.1 µMµM of of Cur-D Cur-D and 3 µµg/mLg/mL of of DRV DRV (Cmax ofof oral oral 400 400 mg mg QD), 1 µµg/mLg/mL of of RTV (Cmax value of oral 100 mg QD) with and withoutmax CSC (10 µg/mL) for 2 days. After 2 daysmax of valuevalue of of oral oral 100 100 mg mg QD) QD) with with and and without without CSC CSC (10 (10 µg/mL)µg/mL) for for 2 2 days. days. After After 2 2 days days of of treatment, p24 levels were measured in the collected media. As expected, both Cur-D and treatment,treatment, p24 p24 levels levels were were measured measured in in the the collected collected media. media. As As expected, expected, both both Cur-D Cur-D and and DRV-RTV showed a significant reduction in p24 levels compared to both control and CSC DRV-RTVDRV-RTV showed showed a a significant signiﬁcant reduction reduction in in p24 p24 levels levels compared compared to to both both control control and and CSC CSC (Figure(Figure(Figure 4).4 4).). Although Although Although notnot not significant, signiﬁcant, significant, the the the reduction reduction reduction in in in viral viral viral load load load with with with Cur-D Cur-D was was slightly slightly lowerlowerlower than thanthan with withwith DRV-RTV. DRV-RTV.DRV-RTV. Thus, Thus, Thus, compared comparedcompared to toto the thethe established establishedestablished ART ARTART regimen, regimen,regimen, Cur-D Cur-DCur-D also alsoalso . waswaswas effective effectiveeffective against againstagainst HIV HIVHIV replication replicationreplication alone alonealone as asas well wellwell asas as inin in thethe the presencepresence presence of of of CSC CSC CSC (Figure (Figure (Figure 44) ).4).

Figure 4. Comparison of the anti-HIV effect of Cur-D with darunavir/ritonavir (DRV/RTV): To FigureFigure 4. 4. ComparisonComparison of ofthe the anti-HIV anti-HIV effect effect of Cur- of Cur-DD with with darunavir/ritonavir darunavir/ritonavir (DRV/RTV): (DRV/RTV): To To compare the anti-HIV effect of Cur-D with DRV/RTV on cigarette smoke condensate (CSC)-in- comparecompare the the anti-HIV anti-HIV effect effect of of Cur-D Cur-D with with DR DRV/RTVV/RTV on on cigarette cigarette smoke condensate (CSC)-induced(CSC)-induced viral replication, differentiated U1 macrophages were treated with Control (DMSO), CSC ducedviral replication, viral replication, differentiated differentiated U1 macrophages U1 macrophages were treated were withtreated Control with (DMSO),Control (DMSO), CSC (10 µCSCg/mL), (10(10 µg/mL), µg/mL), Cur-D Cur-D (0.1 (0.1 µM), µM), and and DRV-RTV DRV-RTV (3 (3 µg/mL/1 µg/mL/1 µg/mL) µg/mL) and and observed observed for for 2 2days. days. HIV HIV viral viral Cur-D (0.1 µM), and DRV-RTV (3 µg/mL/1 µg/mL) and observed for 2 days. HIV viral loads from loadsloads from from media media of of U1 U1 cells cells were were measured measured on on day day 2, 2, using using a ap24 p24 ELISA ELISA kit. kit. One-way One-way ANOVA ANOVA media of U1 cells were measured on day 2, using a p24 ELISA kit. One-way ANOVA with Tukey’s withwith Tukey’s Tukey’s post-hoc post-hoc test test was was applied applied to to comp compareare between between multiple multiple groups. groups. * *and and *** *** represent represent p p

FigureFigureFigure 5. 5.5. Effect Effect of of of Cur-D Cur-D Cur-D on on on CSC-induced CSC-induced CSC-induced HIV HIV HIV replication: replication: replication: Differentiated Differentiated Differentiated U1 U1 U1macrophages macrophages macrophages were were were concomitantlyconcomitantlyconcomitantly treated treated with with CSC CSC CSC (10 (10 (10 µg/mL) µg/mL)µg/mL) and and and Cur-D Cur-D Cur-D (0.1 (0.1 (0.1 µM) µM)µ M)every every every day day dayfor for 3 for 3days. days. 3 days. HIV HIV HIVp24 p24 p24 levelslevelslevels were werewere measured measuredmeasured at at at the the the end end end of of the the treatmenttrea treatmenttment usingusing using anan an ELISAELISA ELISA kit. kit. kit. One-way One-way One-way ANOVA ANOVA ANOVA with with with Tukey’s Tukey’s post-hoc test was applied to compare between multiple groups. *, **, and *** represent p < Tukey’spost-hoc post-hoc test was test applied was applied to compare to compare between between multiple multiple groups. groups. *, **, *, and **, and *** represent*** representp < p 0.05 < , 0.05, p ≤ 0.01, and p ≤ 0.001, respectively when compared to control. ##, and ### represent p ≤ 0.01, 0.05,p ≤ p0.01, ≤ 0.01, and andp ≤ p0.001, ≤ 0.001, respectively respectively when when compared compared to control.to control. ##, ##, and and ### ### represent representp ≤ p 0.01,≤ 0.01, and and p ≤ 0.001, respectively when compared to CSC. andp ≤ p0.001, ≤ 0.001, respectively respectively when when compared compared to CSC.to CSC.

3.5. Cur-D Reduces a Major Pro-Inflammatory Cytokine, IL1β, in HIV Infected Macrophages Cucurbitacins have been shown to exert antioxidant and anti-inflammatory properties [28]. Therefore, we investigated whether the anti-HIV effect of Cur-D is mediated through antioxidant or anti-inflammatory pathways. We observed that a key pro- inflammatory cytokine, IL1β, which is known to play a major role in HIV disease progression [29,30], is significantly increased with CSC exposure (Figure6). Upon Cur-D treatment, Viruses 2021, 13, x 7 of 15 Viruses 2021, 13, x 7 of 15

3.5.3.5. Cur-DCur-D ReducesReduces aa MajorMajor Pro-InflammatoryPro-Inflammatory Cytokine,Cytokine, IL1IL1ββ,, inin HIVHIV InfectInfecteded MacrophagesMacrophages CucurbitacinsCucurbitacins havehave beenbeen shownshown toto exertexert antioxidantantioxidant andand anti-inflammatoryanti-inflammatory proper-proper- Viruses 2021, 13, 1004 7 of 14 tiesties [28].[28]. Therefore,Therefore, wewe investigatedinvestigated whethewhetherr thethe anti-HIVanti-HIV effecteffect ofof Cur-DCur-D isis mediatedmediated throughthrough antioxidantantioxidant oror anti-inflammatoryanti-inflammatory papathways.thways. WeWe observedobserved thatthat aa keykey pro-inflam-pro-inflam- matorymatory cytokine,cytokine, IL1IL1ββ,, whichwhich isis knownknown toto playplay aa majormajor rolerole inin HIVHIV diseasedisease progressionprogression [29,30], is significantly increased with CSC exposure (Figure 6). Upon Cur-D treatment, the[29,30], levels is ofsignificantly IL1β were signiﬁcantlyincreased with decreased. CSC exposure Furthermore, (Figure we 6). observedUpon Cur-D that thetreatment, Cur-D the levels of IL1β were significantly decreased. Furthermore, we observed that the Cur-D treatmentthe levels signiﬁcantlyof IL1β were reduced significantly the levels decreased. of the antioxidantFurthermore, enzyme, we observed SOD1, whilethat the showing Cur-D treatment significantly reduced the levels of the antioxidant enzyme, SOD1, while show- atreatment pattern of significantly reduction in reduced the levels the of levels catalase of the (Figure antioxidant6). enzyme, SOD1, while show- inging aa patternpattern ofof reductionreduction inin thethe levelslevels ofof catalasecatalase (Figure(Figure 6).6).

Figure 6. Effect of Cur-D on IL-1β and antioxidant enzymes (catalase and SOD1): Differentiated FigureFigure 6.6. Effect ofof Cur-DCur-D onon IL-1IL-1β andand antioxidantantioxidant enzymesenzymes (catalase(catalase andand SOD1):SOD1): DifferentiatedDifferentiated U1 U1 macrophages were concomitantly treated with CSC (10 µg/mL) and Cur-D (0.1 µM) continu- macrophagesU1 macrophages were were concomitantly concomitantly treated treated with with CSC CSC (10 (10µg/mL) µg/mL) and and Cur-D Cur-D (0.1 (0.1µM) µM) continuously continuously for 3 days and cells were harvested at the end of the treatment. The expression of IL-1β, anti- forously 3 days for 3 and days cells and were cells harvested were harvested at the endat th ofe theend treatment.of the treatment. The expression The expression of IL-1β of, antioxidantIL-1β, antioxidant enzymes (catalase and SOD1) proteins were measured in differentiated U1 cells (n = 3) by enzymesoxidant enzymes (catalase (catalase and SOD1) and proteins SOD1) proteins were measured were measured in differentiated in differentiated U1 cells U1 (n = cells 3) by (nWestern = 3) by Western blot. One-way ANOVA with Tukey’s post-hoc test was applied to compare between mul- blot.Western One-way blot. One-way ANOVA withANOVA Tukey’s with post-hoc Tukey’s test post-h wasoc applied test was to compareapplied to between compare multiple between groups. multiple groups.*, **, and *** represent p < 0.05 and p < 0.01, p < 0.001, respectively when compared to tiple groups.*, **, and p*** representp p < 0.05 pand p < 0.01, p < 0.001, respectively when compared to *,control. **, and # *** and represent ## represent< 0.05 p < and0.05 and< 0.01, p < 0.01,< 0.001, respectively, respectively when when compared compared to CSC. to control. # and ##control. represent # andp <## 0.05 represent and p

3.6.3.6. Cytotoxicity Cytotoxicity of of CSCCSC andand Cur-DCur-D inin U1U1 CellsCells afterafter CrossingCrossing thethe InInIn VitroVitroVitro MouseMouseMouse BBBBBBBBB Model ModelModel ToTo determine determinedetermine the thethe toxicity toxicitytoxicity of ofof CSC CSCCSC and andand Cur-D Cur-DCur-D across acrossacross the BBB,thethe BBB,BBB, we used wewe usedused mouse mousemouse endothelial endo-endo- andthelialthelial astrocytic andand astrocyticastrocytic cells to cellscells form toto a formform BBB aa layer BBBBBB and layerlayer used andand U1 usedused cells U1U1to cellscells create toto createcreate a modiﬁed aa modifiedmodifiedin vitro inin ® BBBvitrovitro model BBBBBB modelmodel in a Transwell inin aa TranswellTranswell® plate,® asplate,plate, described asas describeddescribed in the Methodsinin thethe MethodsMethods section. section.section. We used WeWe mouse usedused endothelialmousemouse endothelialendothelial and astrocytic andand astrocyticastrocytic cells to cellscells represent toto representrepresent our proposed ourour proposedproposed future futurefuture work workwork using usingusing the HIV thethe miceHIVHIV mice modelmice modelmodel to study toto studystudy the pharmacokinetics thethe pharmacokinetipharmacokineti andcscs pharmacodynamics andand pharmacodynamicspharmacodynamics of Cur-D. ofof Cur-D.Cur-D. The upper TheThe insertupperupper of insertinsert the transwell ofof thethe transwelltranswell system systemsystem had conﬂuent hadhad coconfluentnfluent endothelial endothelialendothelial cells and cellscells the andand lower thethe chamber lowerlower cham-cham- had differentiatedberber hadhad differentiateddifferentiated U1 macrophages. U1U1 macrophages.macrophages. The upper TheThe inserts upperupper were insertsinserts exposed werewere exposedexposed to one dose toto oneone of controldosedose ofof (DMSO),controlcontrol (DMSO),(DMSO), CSC (40 CSCCSCµg/mL), (40(40 µg/mL),µg/mL), and Cur-D andand (0.4 Cur-DCur-DµM), (0.4(0.4 and µM),µM), toxicity andand toxicity wastoxicity measured waswas measuredmeasured every day everyevery for 3dayday days, forfor using 33 days,days, the usingusing LDH thethe assay LDHLDH kit fromassayassay the kitkit culture fromfrom thethe media cultureculture of the mediamedia bottom ofof thethe chamber bottombottom containing chamberchamber U1containingcontaining cells. We U1U1 observed cells.cells. WeWe that observedobserved the treatment thatthat thethe with treatmenttreatment CSC and withwith Cur-D CSCCSC did andand not Cur-DCur-D induce diddid any notnotcellular induceinduce toxicityanyany cellularcellular (Figure toxicitytoxicity7). (Figure(Figure 7).7).

Figure 7. Toxicity of CSC and cur-D in U1 cells after crossing the in vitro blood–brain barrier (BBB) Figure 7. Toxicity of CSC and cur-D in U1 cells after crossing the in vitro blood–brain barrier (BBB) Figuremodel: 7.ToToxicity determine of CSC the toxicity and cur-D of CSC in U1 and cells Cur- afterD across crossing the the BBB,in vitrowe usedblood–brain U1 cells to barrier create (BBB) a model: To determine the toxicity of CSC and Cur-D across the BBB, we used U1 cells to create a model:modified To in determine vitro BBB themodel toxicity in a Transwell of CSC and® plate, Cur-D as acrossdescribed the in BBB, the wemethodology. used U1 cells The to upper create a modified in vitro BBB model in a Transwell® plate, as described in the methodology. The upper modiﬁedinserts containingin vitro BBBendothelial model incells aTranswell were exposed® plate, to a as single described dose of in Control the methodology. (DMSO), CSC The (40 upper inserts containing endothelial cells were exposed to a single dose of Control (DMSO), CSC (40 µg/mL), and Cur-D (0.4 µM), and toxicity from differentiated U1 macrophages present in theµ insertsµg/mL), containing and Cur-D endothelial (0.4 µM), cells and weretoxicity exposed from todifferentiated a single dose U1 of Controlmacrophages (DMSO), present CSC (40in theg/mL), lower wells were measured every day for 3 days, using an LDH assay kit from the culture media andlower Cur-D wells (0.4 wereµM), measured and toxicity every from day differentiated for 3 days, using U1 macrophages an LDH assay present kit from in the the lower culture wells media were of the bottom chamber. One-way ANOVA with Tukey’s post-hoc test was applied to compare measuredof the bottom every chamber. day for 3One-way days, using ANOVA an LDH with assay Tu kitkey’s from post-hoc the culture test mediawas applied of the bottomto compare chamber. between multiple groups One-waybetween multiple ANOVA groups with Tukey’s post-hoc test was applied to compare between multiple groups. 3.7. Treatment with Cur-D Decreases CSC-Induced HIV Replication across the Mouse BBB Model To determine whether Cur-D can reduce the CSC-induced HIV replication in the CNS, we concomitantly treated differentiated U1 cells with one dose of Cur-D (0.4 µM) and CSC (40 µg/mL) across the BBB model. We measured P24 levels every day in the medial collected from U1 cells. Although the effect was variable, we observed that CSC, even in the in vitro BBB model, showed increased viral replication at all the time points Viruses 2021, 13, x 8 of 15

3.7. Treatment with Cur-D Decreases CSC-Induced HIV Replication across the Mouse BBB Model To determine whether Cur-D can reduce the CSC-induced HIV replication in the

Viruses 2021, 13, 1004 CNS, we concomitantly treated differentiated U1 cells with one dose of Cur-D (0.48 µM) of 14 and CSC (40 µg/mL) across the BBB model. We measured P24 levels every day in the medial collected from U1 cells. Although the effect was variable, we observed that CSC, even in the in vitro BBB model, showed increased viral replication at all the time points (day 1–3) (Figure8 8).). Furthermore,Furthermore, wewe observedobserved thatthat Cur-DCur-D couldcould signiﬁcantlysignificantly reducereduce thethe CSC-induced HIV replication in both day 2 and day 3 treatment.

Figure 8. 8. EffectEffect of of CSC CSC and and Cur-D Cur-D on onthe theviral viral load load after after crossing crossing the in the vitroin vitroBBB model:BBB model: To deter- To minedetermine the efficacy the efficacy of Cur-D of Cur-D on CSC-induced on CSC-induced viral viralreplication, replication, we used we used differentiated differentiated U1 cells U1 cells to to ® create a modified modified inin vitro vitro BBB model model in in a a Transwell Transwell® plate,plate, as as described described in in the the methodology. methodology. The The upper inserts containing endothelial cells were exposed to a single dose of Control (DMSO), CSC upper inserts containing endothelial cells were exposed to a single dose of Control (DMSO), CSC (40 µg/mL), and Cur-D (0.4 µM) and observed for 3 days. HIV-1 viral loads from differentiated U1 (40 µg/mL), and Cur-D (0.4 µM) and observed for 3 days. HIV-1 viral loads from differentiated U1 cells were measured every day, using a p24 ELISA kit from the culture media of the bottom chamber.cells One-way were measured ANOVA every with day, Tukey’s using post-hoc a p24 ELISA test kitwas from applied the culture to compare media between of the bottom multiple chamber. groups.One-way * and ANOVA ** represent with Tukey’s p < 0.05post-hoc and p ≤ 0.01, test respectively, was applied when to compare compared between to control. multiple ## and groups. ### *represent and ** represent p ≤ 0.01 pand< 0.05 p ≤ and0.001,p ≤respectively0.01, respectively, when compared when compared to CSC. to control. ## and ### represent p ≤ 0.01 and p ≤ 0.001, respectively when compared to CSC. 3.8. Changes in Pro-Inflammatory and Anti-Inflammatory Cytokines with Exposures of CSC and 3.8. Changes in Pro-Inflammatory and Anti-Inflammatory Cytokines with Exposures of CSC and Cur-D to U1 Cells across the Mouse BBB Model Since the IL-1 β levellevel was was significantly significantly elevated with direct exposure toto CSC,CSC, andand thethe IL1-β levellevel was reduced by Cur-D treatment (Figure6 6),), wewe investigated investigated whetherwhether otherother cytokines and chemokines were also altered. Many cytokines and chemokines have been reported to be altered in HIVHIV andand HIV-positiveHIV-positive smokerssmokers [[9,31].9,31]. Therefore,Therefore, wewe examinedexamined eight cytokines/chemokines cytokines/chemokines across across the the BBB BBB model model (Figure (Figure 9).9 ).As As observed observed with with direct direct exposure,exposure, CSC CSC significantly significantly elevated elevated IL-1 IL-1β whereasβ whereas treatment treatment with with Cur-D Cur-D significantly significantly re- ducedreduced its its level level even even in in the the presence presence of of CSC CSC across across the the BBB BBB model. model. On On the the other hand, treatment with with Cur-D Cur-D elevated elevated IL-6 IL-6 level, level, which which is isknown known to toplay play a dual a dual role role during during in- fectionsinfections [32–35]. [32–35 ].However, However, Cur-D Cur-D did did not not cause cause an an elevation elevation in in IL-6 IL-6 level level in in the presence of CSC. Furthermore, exposure of Cur-D to U1 cells did notnot significantlysignificantly increaseincrease IL-10IL-10 levels comparedcompared toto control control whereas whereas CSC CSC exposure exposu significantlyre significantly reduced reduced it (Figure it (Figure9). Finally, 9). Fi- Viruses 2021, 13, x wenally, did we not did observe not observe a significant a significant difference differen in thece levels in the of levels chemokines of chemokines with either 9with of 15 CSC either or

CSCCur-D or exposureCur-D exposure to U1 cells to U1 (Figure cells9 (Figure). 9).

FigureFigure 9. Measurement 9. Measurement of cytokines of cytokines in in U1-cell U1-cell media media treatedtreated with with CSC CSC and and Cur-D Cur-D acro acrossss the theBBB BBB model: model: The upper Theupper inserts inserts containing endothelial cells were exposed to a single dose of Control (DMSO), CSC (40 µg/mL), and Cur-D (0.4 µM) and containing endothelial cells were exposed to a single dose of Control (DMSO), CSC (40 µg/mL), and Cur-D (0.4 µM) and observed for 3 days. The protein levels of various cytokines and chemokines were measured from the culture media of the observedbottom for chamber 3 days. Thecontaining protein differentiated levels of various U1 macrophages cytokines using and chemokines Human Custom were Procarta measuredplex 8-plex. from theOne-way culture ANOVA media of the bottomwith chamber Tukey’s containing post-hoc test differentiated was applied to U1 co macrophagesmpare between using multiple Human groups. Custom * represents Procartaplex p < 0.05 when 8-plex. compared One-way with ANOVA with Tukey’scontrol. # post-hoc represents test p < was0.05 when applied compared to compare with CSC. between multiple groups. * represents p < 0.05 when compared with control. # represents p < 0.05 when compared with CSC. 3.9. Treatment with Cur-D Decreases CSC-Induced HIV Replication across the Human BBB Model After observing the effect of Cur-D on CSC-induced HIV replication in the mouse BBB model, we further established a human BBB model using human endothelial and astrocyte cell lines. We observed that the human BBB model is relatively more sensitive, and thus the treatment lasted for only two days. Briefly, the upper inserts containing human endothelial cells were exposed to one dose of control (DMSO), CSC (40 µg/mL), Cur- D (0.4 µM), and DRV-RTV (12 µg/mL and 4 µg/mL, respectively) for 2 days. We measured p24 levels in the media collected from U1 cells on Day 2. Although CSC did not show an effect, we observed that both Cur-D and DRV-RTV significantly reduced the viral load compared to control (Figure 10). Importantly, both Cur-D and DRV-RTV reduced CSC- induced HIV replication on Day 2, with DRV-RTV showing a relatively higher effect than Cur-D. Thus, the findings showed a similar effect of Cur-D and DRV-RTV on HIV replication in both the human and mouse BBB models.

Figure 10. Effect of CSC and Cur-D on the viral load after crossing the in vitro human BBB model: To determine the efficacy of Cur-D on CSC-induced viral replication, we used differentiated U1 cells to create a modified in vitro human BBB model in a Transwell® plate, as described in the methodology. The upper inserts containing endothelial cells were exposed to a single dose of Con- trol (DMSO), CSC (40 µg/mL), Cur-D (0.4 µM), and DRV-RTV (12 µg/mL and 4 µg/mL, respectively) and observed for 2 days. HIV viral loads from U1 cells were measured every day, using a p24 ELISA kit from the culture media of the bottom chamber. One-way ANOVA with Tukey’s post-hoc test was applied to compare between multiple groups. *** represents p ≤ 0.001when compared to control. #, and ## represent p < 0.05, and p ≤ 0.01, respectively, when compared to CSC.

Viruses 2021, 13, x 9 of 15

Figure 9. Measurement of cytokines in U1-cell media treated with CSC and Cur-D across the BBB model: The upper inserts containing endothelial cells were exposed to a single dose of Control (DMSO), CSC (40 µg/mL), and Cur-D (0.4 µM) and observed for 3 days. The protein levels of various cytokines and chemokines were measured from the culture media of the bottom chamber containing differentiated U1 macrophages using Human Custom Procartaplex 8-plex. One-way ANOVA with Tukey’s post-hoc test was applied to compare between multiple groups. * represents p < 0.05 when compared with Virusescontrol.2021, 13 ,# 1004 represents p < 0.05 when compared with CSC. 9 of 14

3.9. Treatment with Cur-D Decreases CSC-Induced HIV Replication across the Human BBB Model 3.9. Treatment with Cur-D Decreases CSC-Induced HIV Replication across the Human BBB Model After observing the effect of Cur-D on CSC-induced HIV replication in the mouse After observing the effect of Cur-D on CSC-induced HIV replication in the mouse BBB BBB model, we further established a human BBB model using human endothelial and model, we further established a human BBB model using human endothelial and astrocyte astrocyte cell lines. We observed that the human BBB model is relatively more sensitive, cell lines. We observed that the human BBB model is relatively more sensitive, and thus the and thus the treatment lasted for only two days. Briefly, the upper inserts containing hu- treatment lasted for only two days. Briefly, the upper inserts containing human endothelial man endothelial cells were exposed to one dose of control (DMSO), CSC (40 µg/mL), Cur- cells were exposed to one dose of control (DMSO), CSC (40 µg/mL), Cur-D (0.4 µM), and DRV-RTVD (0.4 µM), (12 andµg/mL DRV-RTV and 4(12µ g/mL,µg/mL respectively)and 4 µg/mL, for respectively) 2 days. We for measured 2 days. We p24 measured levels in thep24 medialevels in collected the media from collected U1 cells from on DayU1 ce 2.lls Although on Day 2. CSC Although did not CSC show did an not effect, show we an observedeffect, we thatobserved both Cur-Dthat both and Cur-D DRV-RTV and significantlyDRV-RTV significantly reduced the reduced viral load the comparedviral load tocompared control (Figureto control 10). (Figure Importantly, 10). Importantly, both Cur-D both and DRV-RTVCur-D and reduced DRV-RTV CSC-induced reduced CSC- HIV replicationinduced HIV on replication Day 2, with on DRV-RTV Day 2, with showing DRV-RTV a relatively showing higher a relatively effect thanhigher Cur-D. effect Thus, than theCur-D. findings Thus, showed the findings a similar showed effect a of similar Cur-D ef andfect DRV-RTV of Cur-D on and HIV DRV-RTV replication on inHIV both repli- the humancation in and both mouse the human BBB models. and mouse BBB models.

Figure 10. Effect of CSCCSC andand Cur-DCur-D onon the the viral viral load load after after crossing crossing the thein in vitro vitrohuman human BBB BBB model: model: To determineTo determine the the efﬁcacy efficacy of Cur-D of Cur-D on CSC-inducedon CSC-induced viral viral replication, replication, we usedwe used differentiated differentiated U1 cells U1 to createcells to a create modiﬁed a modifiedin vitro inhuman vitro BBBhuman model BBB in model a Transwell in a Transwell® plate, as® plate, described as described in the methodology. in the Themethodology. upper inserts The containing upper inserts endothelial containing cells endothelial were exposed cells to were a single exposed dose ofto Controla single (DMSO), dose of Con- CSC trol (DMSO), CSC (40 µg/mL), Cur-D (0.4 µM), and DRV-RTV (12 µg/mL and 4 µg/mL, respec- (40 µg/mL), Cur-D (0.4 µM), and DRV-RTV (12 µg/mL and 4 µg/mL, respectively) and observed tively) and observed for 2 days. HIV viral loads from U1 cells were measured every day, using a for 2 days. HIV viral loads from U1 cells were measured every day, using a p24 ELISA kit from the p24 ELISA kit from the culture media of the bottom chamber. One-way ANOVA with Tukey’s culturepost-hoc media test was of theapplied bottom to compare chamber. between One-way multiple ANOVA groups. with Tukey’s*** represents post-hoc p ≤ 0.001when test was applied com- topared compare to control. between #, and multiple ## represent groups. p < *** 0.05, represents and p ≤ p0.01,≤ 0.001when respectively, compared when compared to control. to #, CSC. and ## represent p < 0.05, and p ≤ 0.01, respectively, when compared to CSC.

4. Discussion

Various chemodietary agents and nutraceuticals have been shown to be effective against several types of cancers and infectious diseases [17–20,36–38]. Among these, curcumin has been tried as adjuvant therapy in clinical studies to treat various conditions largely for cancers [39–41]. Curcumin has also been shown to possess anti-HIV activity [19,20,42]. However, its limited bioavailability impedes its use for therapeutic applications [43,44]. Although cucurbitacins, especially Cur-D, have also been studied for cancer treatments as adjuvant therapy [45–47], their use in the treatment of infectious diseases, especially HIV, is not studied. To the best of our knowledge, this is the first study to show the anti-HIV activity of Cur-D. One of the major limitations of the use of cucurbitacins is its relatively high toxicity profile [17,48,49]. However, the concentrations that we tested (<1-µM range) in the present study were found to be safe (Figure2) and effective in suppressing the HIV replication directly (Figure3) as well as across the BBB model (Figures8 and 10) in differentiated U1 macrophages. Furthermore, the anti-HIV effect of Cur-D was comparable to that of established ART regimen, DRV-RTV, in both the direct as well as BBB model experiments (Figures4 and 10). Thus, our study provides evidence that Cur-D at <1-µM range could be useful in suppressing HIV not only in the peripheral macrophages, but also in the CNS reservoirs such as brain perivascular macrophages and microglia. Cigarette smoking is prevalent among HIV-positive subjects [8,50]. It can cause oxidative damage [9,10], which in turn can induce BBB damage leading to increased BBB permeability [51–53]. The increased BBB permeability augments infiltration of HIV-infected Viruses 2021, 13, 1004 10 of 14

monocytes and influx of other peripheral toxins into the brain [51]. Consequently, it acti- vates neuro-inflammatory pathways by increasing glial activation, leading to HAND [54]. Therefore, it is important to study the effect of Cur-D on CSC-induced HIV replication across the BBB model. We observed that treatment with Cur-D reduces p24 levels across the BBB model, suggesting that it can cross the BBB to a sufficient extent and suppress the HIV replication. Chronic inflammation and immune suppression are the major hallmarks of HIV infection. Dysregulation of cytokine production has been shown to contribute significantly to HIV replication and disease progression [55–57]. Several reports suggest that HIV replication/disease progression is associated with elevated levels of IL-1β [29,30,58]. In the present study, the increased levels of IL-1β with CSC exposure suggest that CSC exacerbates HIV replication. The Cur-D treatment reduces IL-1β levels alone as well as in the presence of CSC exposure, suggesting that its anti-HIV activity is mediated through its anti-inflammatory pathway. Our findings are supported by those of Yang et al. who reported that Cur-D significantly suppressed the production of IL-1β in keratinocytes and thus may be useful as an anti-inflammatory agent for psoriasis [59]. Although Yoshida et al. suggest that Cur-D increases LPS induced Il-1β level, Cur-D alone did not elevate the IL-1β level in macrophages [60]. Interestingly, the IL-6 level was significantly decreased with the CSC exposure. Our results are supported by those of Zhao et al. who reported that CSC exposure significantly reduces the IL-6 secretion in mouse macrophage cell lines [61]. We also observed a similar trend in clinical samples in which the IL-6 level was relatively low in HIV subjects who smoke compared to HIV-positive subjects alone [31]. However, the exact mechanism by which CSC reduces the level of IL-6, a pro-inflammatory cytokine, is not clear. In the current study, treatment with Cur-D showed an increased level of IL-6. A study by Weimer et al. suggests that increased IL-6 secretion together with decreased IL-10 secretion appear to be involved in inducing CD4 helper dysfunction in HIV-positive subjects [62]. In their study, the authors have also observed that a patient who presented with increased IL-6 secretion, but no diminished IL-10 secretion, had a normal T-cell clone helper function. Moreover, the patient did not progress to developing AIDS during a 6-month observation period, despite an extremely low CD4 cell count of 45/µL. This suggests an important role of unaffected IL-10 secretion in a CD4 helper function. In our study, although the treatment with Cur-D increased IL-6 level, it did not significantly affect the IL-10 level, suggesting that increased IL-6 level with Cur-D might not contribute to CD4 cell dysfunction. IL-10 is an important immunoregulatory cytokine with multiple biological effects. In the present study, the IL-10 level was significantly reduced with CSC exposure. These results are in line with our previous findings observed in plasma samples of HIV-positive smokers [31]. Said et al. reported that increased IL-10 production by monocytes is one of the mechanisms by which microbial products inhibit T-cell function in HIV-infected subjects [62]. Furthermore, IL-10 production is positively correlated with increased peripheral CD4+T cell depletion and increased numbers of microbes such as M. tuberculosis in HIV-positive subjects [63]. Overall, these findings suggest a positive correlation of IL-10 production with CD4 T cell dysfunction in HIV infection. In the present study, compared to control, the IL-10 level did not change with Cur-D treatment, suggesting that Cur-D may not cause T-cell dysfunction. To confirm this, we are in the process of developing an HIV-infected T-cell model. The literature and our studies have shown the role of oxidative stress, generated by CSC, on HIV replication [9,10]. As expected, CSC reduced the levels of AOEs, especially SOD1, suggesting an increase in oxidative stress. However, Cur-D alone as well as in the presence of CSC also reduced the level of SOD1. The findings suggest that Cur-D does not suppress HIV, either directly or in the presence of CSC, via the oxidative stress pathway. On the other hand, a decreased level of SOD1 by Cur-D could be explained by its toxic nature, as Cur-D shows toxicity to many cells, especially to cancer cells [45,64,65]. In fact, Viruses 2021, 13, 1004 11 of 14

due to its toxic role to kill cancer cells, Cur-D is studied to be used as adjuvant therapy in cancer treatment [45]. The major limitation of currently used ART drugs is their inability to cross the BBB and eliminate the virus from the brain [66,67]. Some of these ART drugs are also reported to cause neurotoxicity [15]. Thus, there is a need to use chemodietary agents as adjuvant therapy that help suppress the virus in the brain at relatively low and non-toxic ART drug concentration. To achieve the desired effects of ART drugs at low and nontoxic doses, Cur- D can be used as adjuvant therapy in HIV treatment. Although the use of adjuvant therapy is commonly implemented in cancer treatment to reduce the toxicity of chemotherapy, no drug has been tried for HIV treatment. One of the limitations of our study is that we did not show the effect of Cur-D on T-cells, which are the central target of HIV therapies and HIV disease progression. Our goal in this project was to ﬁnd a natural compound that can be used as an adjuvant therapy to suppress viral replication in the brain HIV reservoirs. It is well-documented that the HIV infection of the CNS results from the transmigration of infected CD4+ cells and monocytes across the BBB [68]. However, these HIV-infected cells entering the brain do not constitute an HIV reservoir since they have short half-lives [69,70]. Nevertheless, these cells, especially HIV-infected monocytes, are the source of the infection of three long-lived cell types: astrocytes, perivascular macrophages, and microglial cells. Since HIV infection in astrocytes appears to be non-productive, they cannot serve as HIV reservoirs. Therefore, the evidence suggests that both perivascular macrophages and microglial cells are susceptible to HIV infection and support productive infection. The productive infection in these cells in the brain has been associated with HAND in humans. Therefore, we selected macrophages for the study, as they are the cells of origin for perivascular macrophages and microglia in the CNS. Furthermore, the in vitro BBB models may not mimic the physiological BBB, because they lack three-dimensional structure and endothelial exposure to physiological shear stress, and restrict the maintenance of BBB properties in fully differentiated cells [71]. Therefore, the next goal is to test whether Cur-D can achieve therapeutic concentrations to suppress HIV in the periphery, particularly in T-cells, and in CNS cells. We will also test whether increased Cur-D concentration in the CNS suppresses HIV, as well as enhances the efﬁcacy of ART drugs using both in vitro three-dimensional BBB and in vivo HIV animal models.

5. Conclusions In conclusion, Cur-D reduces HIV replication directly as well as across the BBB models. It is also effective against CSC-induced HIV replication. Therefore, the present study provides the potential for Cur-D to be developed as adjuvant therapy in HIV treatment. It may be used not only to suppress HIV in the brain, but also to reduce the CNS toxicity of currently existing ART drugs. However, before we realize its role as an anti-HIV agent in adjuvant therapy, the detailed pharmacokinetics, tissue distribution, especially in the brain, and pharmacodynamic effects in an appropriate humanized HIV animal model are needed.

Author Contributions: Conceptualization, S.K. (Santosh Kumar) and S.K. (Sunitha Kodidela); Methodology, N.S. and S.K. (Sunitha Kodidela); Formal analysis, S.K. (Sunitha Kodidela), S.K. (Santosh Kumar), and A.K.; writing—original draft preparation, S.K. (Sunitha Kodidela) and S.K. (Santosh Kumar); writing—review and editing, S.K. (Santosh Kumar), A.K. and S.K. (Sunitha Ko- didela); Supervision, S.K. (Santosh Kumar); Funding acquisition, S.K. (Santosh Kumar). All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Institute on Drug Abuse, DA047178. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Viruses 2021, 13, 1004 12 of 14

References 1. Clifford, D.B.; Ances, B.M. HIV-Associated Neurocognitive Disorder (HAND). Lancet Infect. Dis. 2013, 13, 976–986. [CrossRef] 2. Sacktor, N.; Skolasky, R.L.; Seaberg, E.; Munro, C.; Becker, J.T.; Martin, E.; Ragin, A.; Levine, A.; Miller, E. Prevalence of HIV-Associated Neurocognitive Disorders in the Multicenter AIDS Cohort Study. Neurology 2016, 86, 334–340. [CrossRef] 3. Williams, D.W.; Veenstra, M.; Gaskill, P.J.; Morgello, S.; Calderon, T.M.; Berman, J.W. Monocytes Mediate HIV Neuropathogenesis: Mechanisms That Contribute to HIV Associated Neurocognitive Disorders. Curr. HIV Res. 2014, 12, 85–96. [CrossRef] 4. Tozzi, V.; Balestra, P.; Bellagamba, R.; Corpolongo, A.; Salvatori, M.F.; Visco-Comandini, U.; Vlassi, C.; Giulianelli, M.; Galgani, S.; Antinori, A.; et al. Persistence of Neuropsychologic Deficits despite Long-Term Highly Active Antiretroviral Therapy in Patients with HIV-Related Neurocognitive Impairment: Prevalence and Risk Factors. J. Acquir. Immune Defic. Syndr. 2007, 45, 174–182. [CrossRef][PubMed] 5. Edén, A.; Fuchs, D.; Hagberg, L.; Nilsson, S.; Spudich, S.; Svennerholm, B.; Price, R.W.; Gisslén, M. HIV-1 Viral Escape in Cerebrospinal Fluid of Subjects on Suppressive Antiretroviral Treatment. J. Infect. Dis. 2010, 202, 1819–1825. [CrossRef][PubMed] 6. Varatharajan, L.; Thomas, S.A. The Transport of Anti-HIV Drugs across Blood-CNS Interfaces: Summary of Current Knowledge and Recommendations for Further Research. Antivir. Res. 2009, 82, A99–A109. [CrossRef][PubMed] 7. Rao, K.S.; Ghorpade, A.; Labhasetwar, V. Targeting Anti-HIV Drugs to the CNS. Expert Opin. Drug Deliv. 2009, 6, 771–784. [CrossRef][PubMed] 8. Rahmanian, S.; Wewers, M.E.; Koletar, S.; Reynolds, N.; Ferketich, A.; Diaz, P. Cigarette Smoking in the HIV-Infected Population. Proc. Am. Thorac. Soc. 2011, 8, 313–319. [CrossRef] 9. Ande, A.; McArthur, C.; Ayuk, L.; Awasom, C.; Achu, P.N.; Njinda, A.; Sinha, N.; Rao, P.S.S.; Agudelo, M.; Nookala, A.R.; et al. Effect of Mild-to-Moderate Smoking on Viral Load, Cytokines, Oxidative Stress, and Cytochrome P450 Enzymes in HIV-Infected Individuals. PLoS ONE 2015, 10, e0122402. [CrossRef][PubMed] 10. Rao, P.; Ande, A.; Sinha, N.; Kumar, A.; Kumar, S. Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells. PLoS ONE 2016, 11, e0155791. [CrossRef] 11. Ranjit, S.; Sinha, N.; Kodidela, S.; Kumar, S. Benzo(a)Pyrene in Cigarette Smoke Enhances HIV-1 Replication through NF-KB Activation via CYP-Mediated Oxidative Stress Pathway. Sci. Rep. 2018, 8, 10394. [CrossRef] 12. Staying in HIV Care: Other Related Health Issues: Smoking. Available online: https://www.hiv.gov/hiv-basics/staying-in-hiv- care/other-related-health-issues/smoking (accessed on 17 February 2021). 13. Reddy, K.P.; Kong, C.Y.; Hyle, E.P.; Baggett, T.P.; Huang, M.; Parker, R.A.; Paltiel, A.D.; Losina, E.; Weinstein, M.C.; Freedberg, K.A.; et al. Lung Cancer Mortality Associated with Smoking and Smoking Cessation Among People Living with HIV in the United States. JAMA Intern. Med. 2017, 177, 1613–1621. [CrossRef] 14. Rourke, S.B.; Bekele, T.; Rachlis, A.; Kovacs, C.; Brunetta, J.; Gill, M.J.; Carvalhal, A.; Cysique, L.A.; Marcotte, T.; Power, C. Asymptomatic Neurocognitive Impairment is a Risk for Symptomatic Decline over a 3-Year Study Period. AIDS 2021, 35, 63–72. [CrossRef][PubMed] 15. Robertson, K.; Liner, J.; Meeker, R.B. Antiretroviral Neurotoxicity. J. Neurovirol. 2012, 18, 388–399. [CrossRef][PubMed] 16. FDA Approves First Extended-Release, Injectable Drug Regimen for Adults Living with HIV. Available online: https://www.fda. gov/news-events/press-announcements/fda-approves-first-extended-release-injectable-drug-regimen-adults-living-hiv (accessed on 17 February 2021). 17. Kaushik, U.; Aeri, V.; Mir, S.R. Cucurbitacins—An Insight into Medicinal Leads from Nature. Pharm. Rev. 2015, 9, 12–18. [CrossRef] 18. Cai, Y.; Fang, X.; He, C.; Li, P.; Xiao, F.; Wang, Y.; Chen, M. Cucurbitacins: A Systematic Review of the Phytochemistry and Anticancer Activity. Am. J. Chin. Med. 2015, 43, 1331–1350. [CrossRef] 19. Barthelemy, S.; Vergnes, L.; Moynier, M.; Guyot, D.; Labidalle, S.; Bahraoui, E. Curcumin and Curcumin Derivatives Inhibit Tat-Mediated Transactivation of Type 1 Human Immunodeficiency Virus Long Terminal Repeat. Res. Virol 1998, 149, 43–52. [CrossRef] 20. Prasad, S.; Tyagi, A.K. Curcumin and Its Analogues: A Potential Natural Compound against HIV Infection and AIDS. Food Funct. 2015, 6, 3412–3419. [CrossRef] 21. Shah, S.S.A.; Hussain, M.I.; Aslam, M.K.; Rivera, G. Natural Products; Pharmacological Importance of Family Cucurbitaceae: A Brief Review. Mini Rev. Med. Chem. 2014, 14, 694–705. [CrossRef][PubMed] 22. Haque, S.; Kodidela, S.; Sinha, N.; Kumar, P.; Cory, T.J.; Kumar, S. Differential Packaging of Inflammatory Cytokines/Chemokines and Oxidative Stress Modulators in U937 and U1 Macrophages-Derived Extracellular Vesicles upon Exposure to Tobacco Constituents. PLoS ONE 2020, 15, e0233054. [CrossRef] 23. Nicol, M.Q.; Mathys, J.-M.; Pereira, A.; Ollington, K.; Ieong, M.H.; Skolnik, P.R. Human Immunodeficiency Virus Infection Alters Tumor Necrosis Factor Alpha Production via Toll-like Receptor-Dependent Pathways in Alveolar Macrophages and U1 Cells. J. Virol. 2008, 82, 7790–7798. [CrossRef] 24. Cassol, E.; Alfano, M.; Biswas, P.; Poli, G. Monocyte-Derived Macrophages and Myeloid Cell Lines as Targets of HIV-1 Replication and Persistence. J. Leukoc. Biol. 2006, 80, 1018–1030. [CrossRef] 25. Haque, S.; Sinha, N.; Ranjit, S.; Midde, N.M.; Kashanchi, F.; Kumar, S. Monocyte-Derived Exosomes upon Exposure to Cigarette Smoke Condensate Alter Their Characteristics and Show Protective Effect against Cytotoxicity and HIV-1 Replication. Sci. Rep. 2017, 7, 16120. [CrossRef][PubMed] Viruses 2021, 13, 1004 13 of 14

26. Yilmaz, A.; Izadkhashti, A.; Price, R.W.; Mallon, P.W.; de Meulder, M.; Timmerman, P.; Gisslén, M. Darunavir Concentrations in Cerebrospinal Fluid and Blood in HIV-1-Infected Individuals. AIDS Res. Hum. Retrovir. 2009, 25, 457–461. [CrossRef][PubMed] 27. Gong, Y.; Chowdhury, P.; Nagesh, P.K.B.; Rahman, M.A.; Zhi, K.; Yallapu, M.M.; Kumar, S. Novel Elvitegravir Nanoformulation for Drug Delivery across the Blood-Brain Barrier to Achieve HIV-1 Suppression in the CNS Macrophages. Sci. Rep. 2020, 10, 3835. [CrossRef][PubMed] 28. Bernard, S.A.; Olayinka, O.A. Search for a Novel Antioxidant, Anti- Inflammatory/Analgesic or Anti-Proliferative Drug: Cucurbitacins Hold the Ace. J. Med. Plants Res. 2010, 4, 2821–2826. 29. Guo, H.; Gao, J.; Taxman, D.J.; Ting, J.P.Y.; Su, L. HIV-1 Infection Induces Interleukin-1β Production via TLR8 Protein-Dependent and NLRP3 Inflammasome Mechanisms in Human Monocytes. J. Biol. Chem. 2014, 289, 21716–21726. [CrossRef] 30. Feria, M.G.; Taborda, N.A.; Hernandez, J.C.; Rugeles, M.T. HIV Replication is Associated to Inflammasomes Activation, IL-1β, IL-18 and Caspase-1 Expression in GALT and Peripheral Blood. PLoS ONE 2018, 13, e0192845. [CrossRef] 31. Kodidela, S.; Ranjit, S.; Sinha, N.; McArthur, C.; Kumar, A.; Kumar, S. Cytokine Profiling of Exosomes Derived from the Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers. PLoS ONE 2018, 13, e0201144. [CrossRef] 32. Xing, Z.; Gauldie, J.; Cox, G.; Baumann, H.; Jordana, M.; Lei, X.F.; Achong, M.K. IL-6 is an Antiinflammatory Cytokine Required for Controlling Local or Systemic Acute Inflammatory Responses. J. Clin. Investig. 1998, 101, 311–320. [CrossRef] 33. Tanaka, T.; Narazaki, M.; Kishimoto, T. IL-6 in Inflammation, Immunity, and Disease. Cold Spring Harb. Perspect. Biol. 2014, 6. [CrossRef] 34. Fuster, J.J.; Walsh, K. The Good, the Bad, and the Ugly of Interleukin-6 Signaling. EMBO J. 2014, 33, 1425–1427. [CrossRef] [PubMed] 35. Scheller, J.; Chalaris, A.; Schmidt-Arras, D.; Rose-John, S. The Pro- and Anti-Inflammatory Properties of the Cytokine Interleukin-6. Biochim. Biophys. Acta 2011, 1813, 878–888. [CrossRef][PubMed] 36. Cassels, B.K.; Asencio, M. Anti-HIV Activity of Natural Triterpenoids and Hemisynthetic Derivatives 2004–2009. Phytochem. Rev. 2011, 10, 545–564. [CrossRef] 37. Aggarwal, B.B.; Shishodia, S. Molecular Targets of Dietary Agents for Prevention and Therapy of Cancer. Biochem. Pharm. 2006, 71, 1397–1421. [CrossRef] 38. Shanmugam, M.K.; Kannaiyan, R.; Sethi, G. Targeting Cell Signaling and Apoptotic Pathways by Dietary Agents: Role in the Prevention and Treatment of Cancer. Nutr. Cancer 2011, 63, 161–173. [CrossRef] 39. Mansouri, K.; Rasoulpoor, S.; Daneshkhah, A.; Abolfathi, S.; Salari, N.; Mohammadi, M.; Rasoulpoor, S.; Shabani, S. Clinical Effects of Curcumin in Enhancing Cancer Therapy: A Systematic Review. BMC Cancer 2020, 20, 791. [CrossRef] 40. Lotan, Y. A Randomized, Double-Blind, Placebo-Controlled Trial of Curcumin to Prevent Progression of Biopsy Proven, Low-Risk Localized Prostate Cancer Patients Undergoing Active Surveillance. 2020. Available online: https://clinicaltrials.gov/ct2/show/ NCT03769766 (accessed on 25 May 2021). 41. National Center of Oncology, Armenia Study of Efficacy of Curcumin in Combination with Chemotherapy in Patients with Advanced Breast Cancer: Randomized, Double Blind, Placebo Controlled Clinical Trial. 2019. Available online: https:// clinicaltrials.gov/ (accessed on 25 May 2021). 42. Jordan, W.C.; Drew, C.R. Curcumin—A Natural Herb with Anti-HIV Activity. J. Natl. Med. Assoc. 1996, 88, 333. 43. Anand, P.; Kunnumakkara, A.B.; Newman, R.A.; Aggarwal, B.B. Bioavailability of Curcumin: Problems and Promises. Mol. Pharm. 2007, 4, 807–818. [CrossRef][PubMed] 44. Lopresti, A.L. The Problem of Curcumin and Its Bioavailability: Could Its Gastrointestinal Influence Contribute to Its Overall Health-Enhancing Effects? Adv. Nutr. 2018, 9, 41–50. [CrossRef] 45. Ku, J.M.; Hong, S.H.; Kim, H.I.; Lim, Y.S.; Lee, S.J.; Kim, M.; Seo, H.S.; Shin, Y.C.; Ko, S.-G. Cucurbitacin D Exhibits Its Anti-Cancer Effect in Human Breast Cancer Cells by Inhibiting Stat3 and Akt Signaling. Eur. J. Inflamm. 2018, 16.[CrossRef] 46. Ku, J.M.; Kim, S.R.; Hong, S.H.; Choi, H.-S.; Seo, H.S.; Shin, Y.C.; Ko, S.-G. Cucurbitacin D Induces Cell Cycle Arrest and Apoptosis by Inhibiting STAT3 and NF-KB Signaling in Doxorubicin-Resistant Human Breast Carcinoma (MCF7/ADR) Cells. Mol. Cell Biochem. 2015, 409, 33–43. [CrossRef][PubMed] 47. Sikander, M.; Malik, S.; Khan, S.; Kumari, S.; Chauhan, N.; Khan, P.; Halaweish, F.T.; Chauhan, B.; Yallapu, M.M.; Jaggi, M.; et al. Novel Mechanistic Insight into the Anticancer Activity of Cucurbitacin D against Pancreatic Cancer (Cuc D Attenuates Pancreatic Cancer). Cells 2019, 9, 103. [CrossRef] 48. Njoroge, G.N.; Newton, L.E. Edible and Poisonous Species of Cucurbitaceae in the Central Highlands of Kenya. EANH 1994, 83, 101–115. [CrossRef] 49. Blaskovich, M.A.; Sun, J.; Cantor, A.; Turkson, J.; Jove, R.; Sebti, S.M. Discovery of JSI-124 (Cucurbitacin I), a Selective Janus Kinase/Signal Transducer and Activator of Transcription 3 Signaling Pathway Inhibitor with Potent Antitumor Activity against Human and Murine Cancer Cells in Mice. Cancer Res. 2003, 63, 1270–1279. 50. Pacek, L.R.; Latkin, C.; Crum, R.M.; Stuart, E.A.; Knowlton, A.R. Current Cigarette Smoking among HIV-Positive Current and Former Drug Users: Associations with Individual and Social Characteristics. AIDS Behav. 2014, 18, 1368–1377. [CrossRef] 51. Kumar, S.; Rao, P.S.S.; Sinha, N.; Midde, N.M. Chapter 17—Cytochrome P450 and Oxidative Stress as Possible Pathways for Alcohol- and Tobacco-Mediated HIV Pathogenesis and NeuroAIDS. In Neuropathology of Drug Addictions and Substance Misuse; Preedy, V.R., Ed.; Academic Press: San Diego, CA, USA, 2016; pp. 179–188. ISBN 978-0-12-800213-1. Viruses 2021, 13, 1004 14 of 14

52. Sajja, R.K.; Rahman, S.; Cucullo, L. Drugs of Abuse and Blood-Brain Barrier Endothelial Dysfunction: A Focus on the Role of Oxidative Stress. J. Cereb Blood Flow Metab. 2016, 36, 539–554. [CrossRef] 53. Hossain, M.; Sathe, T.; Fazio, V.; Mazzone, P.; Weksler, B.; Janigro, D.; Rapp, E.; Cucullo, L. Tobacco Smoke: A Critical Etiological Factor for Vascular Impairment at the Blood-Brain Barrier. Brain Res. 2009, 1287, 192–205. [CrossRef][PubMed] 54. Chang, L.; Liang, H.; Kandel, S.R.; He, J.J. Independent and Combined Effects of Nicotine or Chronic Tobacco Smoking and HIV on the Brain: A Review of Preclinical and Clinical Studies. J. Neuroimmune Pharmacol. 2020, 15, 658–693. [CrossRef][PubMed] 55. Reuter, M.A.; Pombo, C.; Betts, M.R. Cytokine Production and Dysregulation in HIV Pathogenesis: Lessons for Development of Therapeutics and Vaccines. Cytokine Growth Factor Rev. 2012, 23, 181–191. [CrossRef][PubMed] 56. Mihret, A.; Abebe, M.; Bekele, Y.; Aseffa, A.; Walzl, G.; Howe, R. Impact of HIV Co-Infection on Plasma Level of Cytokines and Chemokines of Pulmonary Tuberculosis Patients. BMC Infect. Dis. 2014, 14, 125. [CrossRef] 57. Freeman, M.L.; Shive, C.L.; Nguyen, T.P.; Younes, S.-A.; Panigrahi, S.; Lederman, M.M. Cytokines and T-Cell Homeostasis in HIV Infection. J. Infect. Dis. 2016, 214 (Suppl. 2), S51–S57. [CrossRef][PubMed] 58. Rollenhagen, C.; Asin, S.N. Enhanced HIV-1 Replication in Ex Vivo Ectocervical Tissues from Post-Menopausal Women Correlates with Increased Inflammatory Responses. Mucosal. Immunol. 2011, 4, 671–681. [CrossRef] 59. Shin, H.M.; Yang, I.J. Cucurbitacin D Inhibited Pro-Inflammatory Cytokine Production and Proliferation in Keratinocytes. Am. J. Ethnomed. 2019.[CrossRef] 60. Song, Y.; Ding, N.; Kanazawa, T.; Yamashita, U.; Yoshida, Y. Cucurbitacin D is a New Inflammasome Activator in Macrophages. Int. Immunopharmacol. 2013, 17, 1044–1050. [CrossRef] 61. Zhao, J.; Li, X.; Xie, F.; Yang, Z.; Pan, X.; Zhu, M.; Shang, P.; Nie, C.; Liu, H.; Xie, J. Immunomodulatory Effects of Cigarette Smoke Condensate in Mouse Macrophage Cell Line. Int. J. Immunopathol. Pharmacol. 2017, 30, 315–321. [CrossRef] 62. Said, E.A.; Dupuy, F.P.; Trautmann, L.; Zhang, Y.; Shi, Y.; El-Far, M.; Hill, B.J.; Noto, A.; Ancuta, P.; Peretz, Y.; et al. Programmed Death-1-Induced Interleukin-10 Production by Monocytes Impairs CD4+ T Cell Activation during HIV Infection. Nat. Med. 2010, 16, 452–459. [CrossRef][PubMed] 63. Diedrich, C.R.; O’Hern, J.; Gutierrez, M.G.; Allie, N.; Papier, P.; Meintjes, G.; Coussens, A.K.; Wainwright, H.; Wilkinson, R.J. Relationship Between HIV Coinfection, Interleukin 10 Production, and Mycobacterium Tuberculosis in Human Lymph Node Granulomas. J. Infect. Dis. 2016, 214, 1309–1318. [CrossRef][PubMed] 64. Takahashi, N.; Yoshida, Y.; Sugiura, T.; Matsuno, K.; Fujino, A.; Yamashita, U. Cucurbitacin D Isolated from Trichosanthes Kirilowii Induces Apoptosis in Human Hepatocellular Carcinoma Cells in Vitro. Int. Immunopharmacol. 2009, 9, 508–513. [CrossRef] 65. Ishii, T.; Kira, N.; Yoshida, T.; Narahara, H. Cucurbitacin D Induces Growth Inhibition, Cell Cycle Arrest, and Apoptosis in Human Endometrial and Ovarian Cancer Cells. Tumour Biol. 2013, 34, 285–291. [CrossRef][PubMed] 66. Ene, L.; Duiculescu, D.; Ruta, S.M. How Much Do Antiretroviral Drugs Penetrate into the Central Nervous System? J. Med. Life 2011, 4, 432–439. [PubMed] 67. Bertrand, L.; Nair, M.; Toborek, M. Solving the Blood-Brain Barrier Challenge for the Effective Treatment of HIV Replication in the Central Nervous System. Curr. Pharm. Des. 2016, 22, 5477–5486. [CrossRef][PubMed] 68. Williams, D.W.; Eugenin, E.A.; Calderon, T.M.; Berman, J.W. Monocyte Maturation, HIV Susceptibility, and Transmigration across the Blood Brain Barrier are Critical in HIV Neuropathogenesis. J. Leukoc. Biol. 2012, 91, 401–415. [CrossRef][PubMed] 69. Zhou, Y.; Zhang, H.; Siliciano, J.D.; Siliciano, R.F. Kinetics of Human Immunodeficiency Virus Type 1 Decay Following Entry into Resting CD4+ T Cells. J. Virol. 2005, 79, 2199–2210. [CrossRef][PubMed] 70. Whitelaw, D.M. Observations on Human Monocyte Kinetics after Pulse Labeling. Cell Tissue Kinet 1972, 5, 311–317. [CrossRef] [PubMed] 71. Sivandzade, F.; Cucullo, L. In-Vitro Blood-Brain Barrier Modeling: A Review of Modern and Fast-Advancing Technologies. J. Cereb. Blood Flow Metab. 2018, 38, 1667–1681. [CrossRef]