International Journal of Molecular Sciences Brief Report Pharmacological Inhibition of IKK to Tackle Latency and Hyperinflammation in Chronic HIV-1 Infection Ifeanyi Jude Ezeonwumelu 1 , Edurne Garcia-Vidal 1 , Eva Riveira-Muñoz 1 , Eudald Felip 1,2,3 , Lucía Gutiérrez-Chamorro 1 , Ignasi Calba 1,2 , Marta Massanella 1,4,5 , Guillem Sirera 6 , Bonaventura Clotet 1,4,5 , Ester Ballana 1,2, * and Roger Badia 1,2, * 1 IrsiCaixa AIDS Research Institute, Hospital Universitari Germans Trias i Pujol, Can Ruti Campus, 08916 Badalona, Spain 2 Health Research Institute Germans Trias i Pujol (IGTP), Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain 3 Medical Oncology Department, Catalan Institute of Oncology, Badalona-Applied Research Group in Oncology (B-ARGO), 08916 Badalona, Spain 4 University of Vic-Central University of Catalonia (UVic-UCC), 08500 Vic, Spain 5 Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), 28029 Madrid, Spain 6 Fight Infections Foundation, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain * Correspondence: eballana@irsicaixa.es (E.B.); rbadia@irsicaixa.es (R.B.) Abstract: HIV latent infection may be associated with disrupted viral RNA sensing, interferon (IFN) signaling, and/or IFN stimulating genes (ISG) activation. Here, we evaluated the use of compounds selectively targeting at the inhibitor of nuclear factor-κB (IκB) kinase (IKK) complex subunits and Citation: Ezeonwumelu, I.J.; related kinases (TBK1) as a novel pathway to reverse HIV-1 latency in latently infected non-clonal Garcia-Vidal, E.; Riveira-Muñoz, E.; lymphoid and myeloid cell in vitro models. IKK inhibitors (IKKis) triggered up to a 1.8-fold increase Felip, E.; Gutiérrez-Chamorro, L.; in HIV reactivation in both, myeloid and lymphoid cell models. The best-in-class IKKis, targeting Calba, I.; Massanella, M.; Sirera, G.; TBK-1 (MRT67307) and IKKβ (TCPA-1) respectively, were also able to significantly induce viral Clotet, B.; Ballana, E.; et al. reactivation in CD4+ T cells from people living with HIV (PLWH) ex vivo. More importantly, Pharmacological Inhibition of IKK to although none of the compounds tested showed antiviral activity, the combination of the distinct Tackle Latency and Hyperinflammation IKKis with ART did not affect the latency reactivation nor blockade of HIV infection by ART. Finally, in Chronic HIV-1 Infection. Int. J. Mol. Sci. 2022, 23, 15000. https://doi.org/ as expected, IKKis did not upregulate cell activation markers in primary lymphocytes and innate 10.3390/ijms232315000 immune signaling was blocked, resulting in downregulation of inflammatory cytokines. Overall, our results support a dual role of IKKis as immune modulators being able to tackle the HIV latent Academic Editors: Asia reservoir in lymphoid and myeloid cellular models and putatively control the hyperinflammatory Fernandez Carvajal and responses in chronic HIV-1 infection. Gerard Pujadas Received: 2 November 2022 Keywords: HIV-1; latency reversing agents; IKK; TANK binding kinase-1 Accepted: 28 November 2022 Published: 30 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- The human immunodeficiency virus (HIV) reservoir is established early after HIV iations. infection through a complex and multifactorial process [1]. Although several latency reversing strategies have been proposed, none have provided clinical benefits so far. New strategies targeting the integrated HIV provirus, either directed to latency reversal or to the permanent silencing of HIV are, therefore, one of the major research priorities to HIV Copyright: © 2022 by the authors. cure [2,3]. The HIV reservoir comprises different lymphocyte T populations [4] but also Licensee MDPI, Basel, Switzerland. other less studied cell types from the myeloid lineage. Understanding the contribution of This article is an open access article myeloid cells to HIV persistence is also necessary to achieve a complete HIV remission [5,6]. distributed under the terms and Innate immunity plays an important role in the early control of HIV infection, and conditions of the Creative Commons cumulative evidence suggests a link between functional innate immunity and interferon Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ (IFN)-mediated responses and the formation, composition, and long-term maintenance 4.0/). of the HIV reservoir [7]. HIV-1 transcription is a multifactorial process that requires the Int. J. Mol. Sci. 2022, 23, 15000. https://doi.org/10.3390/ijms232315000 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 15000 2 of 11 recruitment to the nucleus of nuclear factor-κB (NF-κB) transcription factor, among others, to initiate viral transcription [8,9]. In this process, NF-κB phosphorylation is mediated by the inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex, which is tightly regulated through the specific binding of distinct regulatory factors. The IKK family is a master regulator of many biological processes, including cell growth, metabolism, apoptosis, or cell cycle [10–13]. The NF-κB pathway and its regulatory counterparts play a complex role during the replication of primate lentiviruses. NF-κB is essential for the induction of efficient proviral gene expression, but also contributes to the innate immune response and induces the expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and, by suppressing it at later stages to minimize antiviral gene expression [14]. IKK-related kinases Tank binding kinase 1 (TBK1) and IκB kinase-ε (IKKε) are confluent downstream regulators of pattern recognition receptors (PRRs) signaling pathways involved in the innate sensing of foreign nucleic acids. Therefore, NF-κB-IKK interplay becomes a key target for viruses (i.e., HIV), which have evolved mechanisms to modulate the NF-κB–IkB-IKK crosstalk to favor their replication cycle [15]. Currently, several inhibitors of IKK complex and the IKK-related kinases are under development for the treatment of cancer and inflammatory diseases [14,16–18]. Taking advantage of our previous experience in the screening of compound libraries [19–21], we explored the role of IKK inhibitors (IKKis) as potential latency reversing agents (LRAs) in the context of HIV chronic infections. 2. Results 2.1. IKKis Induce HIV Reactivation in Distinct Models of HIV Latency In Vitro To assess the latency reactivation capacity of IKKis, we evaluated their role in lymphoid and myeloid models of HIV latency (summarized in Supplementary Table S1). The LRA activity of selected IKKis was evaluated according to their highest subtoxic concentration for each specific compound of interest. Since toxicity is a major concern for the use of histone deacetylase inhibitors (HDACis), we used them at subtoxic concentrations as previously described [22]. Compared to HDACis, IKKis showed low cytotoxicity at micromolar level in the J-HIG and HL-HIG latency models (Figure 1A,B). Six of the tested inhibitors were able to induce viral reactivation in both lymphoid and myeloid cell models (Figure 1A,B), without significantly compromising cell viability with the exception of INH2 at 20 µM in the myeloid model. HIV-1 latency reversing activity of IKKis at the micromolar level was comparable to subtoxic concentrations of HDACi of panobinostat (PNB) and vorinostat (VOR) in both models. These results were confirmed in latently infected ACH-2 cells by measuring HIV p24 Ag in the supernatant (Supplementary Figure S1). The selective TBK/IKKεi MRT67307 is an improved version of BX-795 with less off- targets described effects [12] and TPCA-1 is a selective IKKβi, both representing a 1.6- and 1.8-fold viral reactivation increase at the highest concentrations tested (respectively p < 0.01, compared to the non-treated control). Therefore, they were selected as representative IKKis for further characterization of their LRA potential. 2.2. TBK1/IKKε and IKKβ Inhibitors Induce HIV Reactivation Ex Vivo in CD4+ T Cells from HIV+ Individuals Viral reactivation capacity of the representative IKK inhibitors was evaluated ex vivo in CD4+ T cells from HIV+ individuals (n = 3, Supplementary Table S2). Both IKKis, MRT67307 and TPCA-1, induced HIV-1 reactivation in all participant samples similar to the positive control phorbol-12-myristate-13-acetate (PMA)/ionomycin. Despite the variability in HIV-RNA abundance between samples, HIV-1 reactivation was observed in all patients for MRT67307 (2.1–2.2-fold induction) and TPCA1 (1.3–3.5-fold change) compared to untreated cells (Figure 1C). Int. J. Mol. Sci. 2022, 23, 15000 3 of 11 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 11 Figure 1. Figure 1. IKK Inhibitors IKK Inhibitors induceinduce HIV reactivation HIV reactivation in ininvitro in vitro andand ex vivo ex vivo modelsofoflatency. models latency. (A) (A) HIV HIV reactivation induced by IKKis in latently infected lymphoid Jurkat (J-HIG) and (B) myeloid HL-60 reactivation induced by IKKis in latently infected lymphoid Jurkat (J-HIG) and (B) myeloid HL-60 (HL-HIG) cells. The activity of IKKis MRT67307, TPCA-1, BX795, Inh 1, Inh2, GSK8612 and SC514 (HL-HIG)was cells. The activity determined by theofquantification IKKis MRT67307, of GFP+TPCA-1, cells (%)BX795, Inh 1, Inh2, after culturing J-HIGGSK8612 or HL-HIG and SC514 cells with was determined IKKis forby24the quantification h. HDCAi of GFP+ panobinostat (PNB)cells and(%) after culturing vorinostat (VOR) were J-HIGusedorasHL-HIG controls cells with for HIV-1 IKKis for reactivation. 24 h. HDCAi Basal reactivation (PNB) panobinostat (grey-dashed line) in J-HIG and vorinostat and HL-HIG (VOR) were used wasas established according controls for HIV-1 to the no-drug condition (ND). Toxicity of IKKis (red-dashed line) in J-HIG and HL-HIG cells was reactivation. Basal reactivation (grey-dashed line) in J-HIG and HL-HIG was established according measured as percentage of viable cells (LIVE/DEAD™ staining) by flow cytometry related to the to the no-drug condition ND condition. (C)(ND). Ex vivoToxicity of IKKis (red-dashed viral reactivation capacity of IKKisline) inin J-HIG CD4+ andfrom T cells HL-HIG HIV+ cells was individu- measuredals.as percentage HIV-RNA copies of viable werecells (LIVE/DEAD™ determined staining) in the cell culture by flow cytometry supernatant from HIV+ related CD4+ T to theincu- cells ND condition.bated (C) Exfor vivo 72 h with viral4reactivation µM of MRT67307 or TPCA-1. capacity of IKKis PMA/ionomycin in CD4+ T cells (50 from ng/mL/1 HIV+µM,individuals. PMAi) was HIV-RNAused copiesas the positive control of HIV-1 reactivation. Fold-change (FC) relative to ND are indicated were determined in the cell culture supernatant from HIV+ CD4+ T cells incubated above each bar for the respective treatment conditions. FC values, if not available (na) because of for 72 h with 4 µM undetermined of MRT67307 RT-qPCR ctorvalues TPCA-1.(UD),PMA/ionomycin are highlighted in (50 ng/mL/1 the chart. PMAi) wascapacity µM, reactivation (D) HIV-1 used as the positive control of IKKis of HIV-1 in the presencereactivation. Fold-change of antiretroviral (FC) relative drugs. Reactivation toof ability ND are indicated MRT67307 (4 µM)above and TPCA- each bar for the1 respective (4 µM) is nottreatment affected by the ART (efavirenz, conditions. FC values, EFVifat 0.3available not µM or raltegravir, (na) becauseRAL atof2.2 µM), neither undetermined RT-qPCR in ctlatently values infected (UD), are lymphoid J-HIG (left highlighted panel) in the nor(D) chart. myeloid HIV-1 HL-HIG cells (right reactivation panel).ofViral capacity IKKisreac- in tivation was measured 24 h after incubation with the corresponding conditions. The grey-dashed the presence of antiretroviral drugs. Reactivation ability of MRT67307 (4 µM) and TPCA-1 (4 µM) is line indicates reactivation in the PMAi control. Red dots indicate relative cell viability. Mean ± SD not affected by the of three ART (efavirenz, independent EFV at experiments 0.3 µM*or is shown. p 1 year. HIV-RNA levels were >110,000 viral load (VL) pre-ART copies/mL, and <40 copies/mL during for at least 2 years at study en- try. The immunological and virological characteristics from all participants are found in supplementary Table S2. 4.2. Compounds MRT67306 and BX795 were purchased from Invivogen (Ibian Technologies, Zaragoza, Spain). TCPA-1 (S2824), TBK1/IKK-IN-1 (S8922), TBK1/IKK-IN-2 (S0425), SC514 (S4907), and GSK8612 (S8872) were purchased from Selleckchem (Munich, Germany). Vorinostat was purchased from Prochifar srl (Lombardy, Italy), and panobinostat was purchased from LC Laboratories. Antiretroviral compounds raltegravir (RAL) and efavirenz (EFV) as well as lipopolisaccaride (LPS) and PMA were obtained from Sigma-Merck, Burlington, MA, USA. All compounds were reconstituted in dimethyl sulfoxide (DMSO) and stored at −20 ◦ C until use. 4.3. Virus Envelope-deficient HIV-1 NL4-3 clone encoding IRES-GFP (NL4-3-GFP) was pseu- dotyped with VSV-G by cotransfection of HEK293T cells using polyethylenimine (Poly- sciences, Warrington, PA, USA) as previously described [34,35]. Three days after trans- fection, supernatants were harvested, filtered, and stored at −80 ◦ C. Viral stocks were concentrated using Lenti-X concentrator (Clontech, Mountain View, CA, USA). Viruses were titrated by infection of Jurkat or HL-60 cells followed by GFP quantification by flow cytometry. 4.4. Generation of Latently Infected Cells Latently infected Jurkat (J-HIG) and HL-60 (HL-HIG) cells were generated by following a modification of the protocol described by Li et al. [36,37]. Briefly, cells were generated after acute infection of CD4+ Jurkat or HL-60 cells with VSV-pseudotyped HIV-1 NL4- 3-GFP and were maintained in culture for 10 days to allow the attrition of productively infected cells. 4.5. HIV Reactivation In Vitro in Latently Infected Cells HIV reactivation was measured as described previously [37]. Briefly, J-HIG or HL-HIG cells were incubated for 24 h with different concentrations of IKKIs. HDACi PNB and VOR were used as controls for HIV-1 reactivation. Reactivation of HIV was monitored as the percentage of living GFP+ cells by flow cytometry. Similarly, ACH-2 cells were cultured in the presence or absence of IKKis, PNB, or VOR for 48 h at 37 ◦ C and 5% CO2 . HIV reactivation was measured by the production of HIV CA p24 antigen in the supernatant using Genscreen HIV-1 Ag ELISA (BioRad, Hercules, CA, USA) according to manufacturer’s instructions. Int. J. Mol. Sci. 2022, 23, 15000 8 of 11 4.6. Measurement of HIV Production in CD4+ T Cells from HIV+ Individuals To assess HIV-1 reactivation, CD4+ T cells from HIV+ individuals were pre-incubated with 10 µM of pan-caspase inhibitor Q-VP-Oph (Merck, Ref. SML0063) for 2 h at 37 ◦ C and 5% CO2 . Then, 3.5–5 × 105 cells were plated/treatment condition with the LRAs compounds/controls. The medium was supplemented with recombinant IL-2 (rIL-2) (30 IU/mL; Roche) and antiretroviral drugs RAL (2.2 µM) and EFV (0.3 µM) to avoid multiple cycles of HIV reinfection. Sample processing and HIV production measurement by ultrasensitive semi-nested RT qPCR were performed as previously described [21,38]. 4.7. Quantitative RT-Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted using NucleoSpin RNA II kit (Ref. 740955, Macherey- Nagel, Düren, Germany) as recommended by the manufacturer. Reverse transcription was performed using the PrimeScript™ RT-PCR Kit (RR036A, Takara, Kusatsu, Japan) following the manufacturer’s instructions. mRNA levels of all genes were measured by two-step quantitative RT-PCR and normalized to GAPDH mRNA expression using the DDCt method. The following primers and DNA probes were obtained from TaqMan Gene expression assays (Thermofisher Scientific, Waltham, MA, USA): GAPDH (Hs00266705_g1), CXCL10 (Hs00171042_m1), IL-1β (Hs01555410_m1), IL8 (Hs00174103_m1), and TNFα (Hs00174128_m1). 4.8. Western Blot The treated cells were rinsed, lysed, subjected to SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane, as previously described [37]. The follow- ing antibodies were used for immunoblotting: anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000; Pierce, Dallas, TX, USA); anti-GAPDH (1:2500; ab9485; Abcam, Cambridge, UK); anti-phosphoSTAT1 (1:1000; 9167, Cell Signalling, Danvers, MA, USA), anti-MDA5 (1:500; 5321, Cell Signalling). Blots were immersed in chemiluminescent substrate (SuperSignal West Pico Plus or Femto, Thermo Fisher Sci- entific), and the signal was visualized using ChemiDoc MP imaging system (BIORAD, Hercules, CA, USA). 4.9. Flow Cytometry Cells were labeled with the following anti-human antibodies: CD3 FITC (345763), HLA-DR PE-Cy™7 (335830) purchased from BD and CD4 BV786 (317442), CD25 APC (302610), and CD69 BV650 (310934), all obtained from Biolegend (Palex, Spain). Cells were incubated for 40 min at room temperature in the dark. Then, cells were washed with phosphate buffered saline (PBS) and fixed with 1% formaldehyde (FA). For intracellular assessment of cytokine production, 1 × 105 PBMCs/well were seeded in a polypropylene round-bottom 96-well plate in the presence of the compounds. Two hours after, protein transport inhibitors GolgiPlug (5 µL/mL) and GolgiSTOP (0.66 µL/mL) (Becton Dickinson, Franklin Lakes, NJ, USA) were added and incubated overnight. Cells were then washed with PBS and incubated at 4 ◦ C with BD Cytofix/Cytoperm (Becton Dickinson, USA) for 45 min. Afterwards, cells were washed twice with BD Perm/Wash (Becton Dickinson, USA) diluted 1:10 in water, incubated at 4 ◦ C with the following antibodies (all from BioLegend, Palex, Spain): PE anti-human IL-6 (501107), FITC anti- human IL-1β (511705), Brilliant Violet 650 anti-human TNF-α (502938) (1:50 in diluted Perm/Wash) for 1 h, washed twice again in diluted Perm/Wash and resuspended in formaldehyde 1% in PBS. Cells were analyzed by flow cytometry (BD FACSCelesta™ cell analyzer, BD, Franklin Lakes, NJ, USA) on the same day. Flow cytometry was performed in a FACS LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using the FlowJo™ Software Version 10.6.1. (Becton Dickinson, 2019). Analysis of the global immune profile of PBMCs was done using OMIQ data analysis software (www.omiq.ai, last access on 24 September 2022). Cells were gated on CD3+CD4+ singlets and individual files were concatenated and clustered as Int. J. Mol. Sci. 2022, 23, 15000 9 of 11 a whole in each treatment condition using the optimized t-distributed stochastic neighbor embedding (opt-SNE) algorithm for dimensional reduction and visualization. 4.10. Evaluation of Cytotoxicity Cells were treated at the indicated doses of the test compounds between 24 to 72 h (according to each specific experiment) and then stained for 30 min with LIVE/DEADTM Fixable Green Dead Cell Stain Kit (Invitrogen, Waltham, MA, USA, Thermofisher Scien- tific) in PBS according to the manufacturer’s instructions. Alternatively, viable cells were identified according to forward and side laser light scatter flow cytometry analysis, as described [39]. 4.11. Anti-HIV Assays Antiviral activity of TBK1 and IKKβ inhibitors was assessed on Jurkat and HL-60 cells infected with pseudotyped HIV-1, expressing a GFP reporter gene (VSV-HIG). Untreated condition and antiretroviral compounds RAL and EFV were included as controls. Antiviral activity was measured 48 h after infection as measured as the expression of GFP by flow cytometry. Determinations were performed in triplicates and data calculated from three independent experiments. 4.12. Statistical Methods Data were analyzed with the PRISM statistical package. If not stated otherwise, all data were normally distributed and expressed as mean ± SD. p-values were calculated using an unpaired, two-tailed, t-student test. Supplementary Materials: The following material is available online at https://www.mdpi.com/ article/10.3390/ijms232315000/s1. References [12,40–44] are cited in the supplementary materials. Author Contributions: Conceptualization, I.J.E., E.B. and R.B.; methodology, I.J.E., E.R.-M. and R.B.; validation, I.J.E., E.B. and R.B.; formal analysis, I.J.E., E.G.-V., I.C., E.R.-M., E.F., L.G.-C., E.B. and R.B.; investigation, I.J.E., E.G.-V., I.C., E.R.-M., E.F., L.G.-C., E.B. and R.B.; resources, G.S. and M.M.; writing—original draft preparation, I.J.E., MM, E.B. and R.B.; writing—review and editing, I.J.E., B.C., M.M., E.B. and R.B.; supervision, E.B. and R.B.; funding acquisition, B.C., E.B. and R.B. All authors have read and agreed to the published version of the manuscript. Funding: This study has been funded by Instituto de Salud Carlos III, Fondo de Investigación Sanitaria (FIS) through the project PI19/00194 (co-funded by European Regional Development Fund/European Social Fund) “Investing in your future”) and the NeCeTAR project from GRIFOLS. IE is a research fellow from la Caixa foundation (LCF/BQ/IN18/11660017) co-funded by the Euro- pean Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 713673. EGV (SLT017/20/000090, PERIS, Departament de Salut) and LGC (2019 FI-B00420 AGAUR, Secretaria d’Universitats i Recerca) are research fellows from Generali- tat de Catalunya and Fons Social Europeu. EF, RB, and EB are research fellows from ISCIII-FIS (CM20/00027; CP19/00011 and CPII19/00012, respectively). MM was granted with RYC2020- 028934-I/AEI/10.13039/501100011033 from the Spanish Ministry of Science and Innovation and State Research Agency, MCIN/AEI/10.13039/501100011033 and the European Social Fund “investing in your future”. Institutional Review Board Statement: The study was conducted according to the guidelines and regulations of the Declaration of Helsinki and approved by the Scientific Committee of Institut de Recerca de la Sida—IrsiCaixa and the Ethics Review Board of Hospital Germans Trias i Pujol (CEI PI-21-119). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We thank Foundation Dormeur for the financial support for the acquisition of the QuantStudio-5 real time PCR system. Int. J. Mol. Sci. 2022, 23, 15000 10 of 11 Conflicts of Interest: The authors declare no conflict of interest. References 1. García, M.; Buzón, M.J.; Benito, J.M.; Rallón, N. Peering into the HIV reservoir. Rev. Med. Virol. 2018, 28, e1981. [CrossRef] [PubMed] 2. 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