Pharmacological Inhibition of IKK to Tackle Latency and Hyperinflammation in Chronic HIV-1 Infection

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 related kinases (TBK1) as a novel pathway to reverse HIV-1 latency in latently infected non-clonal lymphoid and myeloid cell in vitro models. IKK inhibitors (IKKis) triggered up to a 1.8-fold increase in HIV reactivation in both, myeloid and lymphoid cell models. The best-in-class IKKis, targeting TBK-1 (MRT67307) and IKKβ (TCPA-1) respectively, were also able to significantly induce viral reactivation in CD4+ T cells from people living with HIV (PLWH) ex vivo. More importantly, although none of the compounds tested showed antiviral activity, the combination of the distinct IKKis with ART did not affect the latency reactivation nor blockade of HIV infection by ART. Finally, as expected, IKKis did not upregulate cell activation markers in primary lymphocytes and innate 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 reservoir in lymphoid and myeloid cellular models and putatively control the hyperinflammatory responses in chronic HIV-1 infection.


Introduction
The human immunodeficiency virus (HIV) reservoir is established early after HIV 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 cure [2,3]. The HIV reservoir comprises different lymphocyte T populations [4] but also other less studied cell types from the myeloid lineage. Understanding the contribution of myeloid cells to HIV persistence is also necessary to achieve a complete HIV remission [5,6].
Innate immunity plays an important role in the early control of HIV infection, and cumulative evidence suggests a link between functional innate immunity and interferon (IFN)-mediated responses and the formation, composition, and long-term maintenance of the HIV reservoir [7]. HIV-1 transcription is a multifactorial process that requires the 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][11][12][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][17][18]. Taking advantage of our previous experience in the screening of compound libraries [19][20][21], we explored the role of IKK inhibitors (IKKis) as potential latency reversing agents (LRAs) in the context of HIV chronic infections.

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 offtargets 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.

Antiretroviral Drugs Do Not Interfere with HIV Reactivation Activity Induced by IKKis
In clinical settings, LRAs have to be combined with ART to avoid new rounds of infection. IKKis showed no antiviral activity in in vitro models (Supplementary Figure S2). To evaluate the possibility of administering IKKis with ART, we evaluated the potential interaction between MRT67307, TPCA-1, and the RT inhibitor efavirenz (EFV) and the integrase inhibitor raltegravir (RAL) at concentrations where EFV and RAL harbor anti-HIV activity [23]. Neither EFV nor RAL modified the IKKis LRA activity in J-HIG and HL-HIG ( Figure 1D). Accordingly, IKKis did not modify the anti-HIV activity of ART (Supplementary Figure S3). Therefore, studied IKKis did not show any synergistic or antagonistic effect when combined with antiretroviral drug and their reactivation capacity was neither modified, indicating the feasibility of combinations with the current antiretroviral regimens.

IKKis Do Not Modify Cell Activation in Ex Vivo Treated Primary CD4+ T Cells
The effect of IKKis on cell activation and innate immune signaling was also evaluated. Interestingly, treatment with MRT67307 or TPCA-1 blocked cytokine expression of IL1-β, IL8, CXCL10 and TNFα (30% to 80% reduction depending on each cytokine; p < 0.05 and p < 0.001 respectively), in contrast to the PMA control ( Figure 2A). Similarly, treatment of peripheral blood mononuclear cells (PBMCs) with MRT67307 or TPCA-1 did not trigger pro-inflammatory cytokine production (IL1-β, IL-6, TNFα), contrary to PMAi or LPS, which significantly induced the expression of IL-1β (LPS p < 0.001), IL-6 (LPS p < 0.01) and TNFα (LPS p < 0.05; PMAi p < 0.001) compared to the ND control at subtoxic concentrations ( Figure 2B). Evaluation of the innate immune signaling activation confirmed the reported effects on targeted pathways, characterized by decreased phosphorylation of STAT1 together with decreased expression of melanoma differentiation-associated gene 5 (MDA5), although results for this intracellular RNA sensor belonging to the Retinoic acid-inducible gene I-like receptors (RLRs) family were statistically non-significant ( Figure 2C). Moreover, IKKis did not promote the expression of cell surface activation markers CD25, CD69, and HLA-DR ex vivo in CD4+ T cells from HIV-negative donors ( Figure 2D,E). Indeed, TPCA-1 treatment reduced the expression of CD25 (72% decrease, p < 0.001) and HLA-DR (30% decrease, p < 0.05).

Antiretroviral Drugs Do Not Interfere with HIV Reactivation Activity Induced by IKKis
In clinical settings, LRAs have to be combined with ART to avoid new rounds of infection. IKKis showed no antiviral activity in in vitro models (Supplementary Figure S2). To evaluate the possibility of administering IKKis with ART, we evaluated the potential interaction between MRT67307, TPCA-1, and the RT inhibitor efavirenz (EFV) and the integrase inhibitor raltegravir (RAL) at concentrations where EFV and RAL harbor anti-HIV activity [23]. Neither EFV nor RAL modified the IKKis LRA activity in J-HIG and HL-HIG ( Figure  1D). Accordingly, IKKis did not modify the anti-HIV activity of ART (Supplementary Figure  S3). Therefore, studied IKKis did not show any synergistic or antagonistic effect when combined with antiretroviral drug and their reactivation capacity was neither modified, indicating the feasibility of combinations with the current antiretroviral regimens.

IKKis Do Not Modify Cell Activation in Ex Vivo Treated Primary CD4+ T Cells
The effect of IKKis on cell activation and innate immune signaling was also evaluated. Interestingly, treatment with MRT67307 or TPCA-1 blocked cytokine expression of IL1-β, IL8, CXCL10 and TNFα (30% to 80% reduction depending on each cytokine; p < 0.05 and p < 0.001 respectively), in contrast to the PMA control ( Figure 2A). Similarly, treatment of peripheral blood mononuclear cells (PBMCs) with MRT67307 or TPCA-1 did not trigger pro-inflammatory cytokine production (IL1-β, IL-6, TNFα), contrary to PMAi or LPS, which significantly induced the expression of IL-1β (LPS p < 0.001), IL-6 (LPS p < 0.01) and TNFα (LPS p < 0.05; PMAi p < 0.001) compared to the ND control at subtoxic concentrations ( Figure 2B). Evaluation of the innate immune signaling activation confirmed the reported effects on targeted pathways, characterized by decreased phosphorylation of STAT1 together with decreased expression of melanoma differentiation-associated gene 5 (MDA5), although results for this intracellular RNA sensor belonging to the Retinoic acidinducible gene I-like receptors (RLRs) family were statistically non-significant ( Figure 2C). Moreover, IKKis did not promote the expression of cell surface activation markers CD25, CD69, and HLA-DR ex vivo in CD4+ T cells from HIV-negative donors ( Figure 2D,E). Indeed, TPCA-1 treatment reduced the expression of CD25 (72% decrease, p < 0.001) and HLA-DR (30% decrease, p < 0.05).

Discussion
The IKK-related kinases TBK1 and IKKε play prominent roles in mediating IFN production upon pathogen recognition, leading to activation of relevant innate immune mediators, including transcription factors, IFN stimulating genes (ISGs), and induction of antiviral IFN-JAK-STAT signaling pathway, being, therefore, putative therapeutic targets for modulating innate immunity.
Innate immunity in HIV-1 pathogenesis is best understood in the context of acute infection. However, recent data suggest that innate immunity can also be used to improve the efficacy of HIV-1 cure strategies [24]. Here, we characterize the latency reactivation capacity of commercially available IKKis. In our study, we postulate that regulation of IKK-TBK inhibition may also represent a novel therapeutic intervention to revert HIV latency, which also has the potential to avoid the unwanted autoimmune-and or inflammation-related side effects associated with latency-reversing strategies. We demonstrate that IKKis have the capacity to reverse HIV-1 latency in lymphoid and myeloid cellular models. Accordingly, NF-κB modulation, through siRNA silencing of IkBα and NF-κB binding to the kB sites of the HIV-LTR, was previously described to activate HIV in latently infected monocytic U1 and lymphoid J-Lat 10.6 [10,25]. These results were confirmed in ex vivo CD4+ T cells from HIV+ individuals by ultra-sensitive semi-nested qPCR that revealed high IKKis-induced viral transcription comparable to PMA and ionomycin reactivation, as previously reported [26]. These results indicate the importance of the inhibition of the TBK-IKK, as key regulators of NF-κB and interferon regulatory factors 3 and 7 (IRF-3/IRF-7) [14] in the context of HIV latency. Our data are in clear contrast to the well-described role of NF-κB as a transcription factor affecting HIV-1 transcription. However, several evidences from the literature support the idea that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB [27]. Moreover, other transcription factors have also been demonstrated to participate in HIV reactivation, resulting in a far more complicated process than the oversimplified LTR-driven transcription by NF-κB [28,29]. Finally, compensatory IKK regulatory mechanisms cannot be excluded [12]. An in-depth description of the underlying mechanisms controlling IKKis capacity as LRA is warranted in future studies.
Novel LRAs must induce the transcription of latent HIV without triggering global cell immune activation or chronic inflammation [30]. In one of the earliest studies investigating the potency of TLR agonists as LRAs, Novis et al. reported that the TLR1/2 agonist Pam3CSK4 reactivates HIV-1 transcription via NF-κB and NFAT-dependent pathways, but without induction of IFNs or T cell activation [31]. Although Pam3CSK4 showed less potency compared to the HDACi panobinostat, this finding lends credence to the possibility of harnessing immune modulation as a strategy to reverse HIV-1 latency and circumvent immune cell activation. The modulation of NF-κB by cytokines/chemokines or PKC agonists has been discouraged due to drug tolerability concerns [25,32,33]. Alternatively, the LRA activity of IKKis had minimal impact on cell activation markers in primary CD4+ T cells. Importantly, IKKi-induced HIV reactivation was accompanied with a partial block of the innate immune signaling and decreased induction of proinflammatory cytokines such as IL8, IL1β, and the chemokine CXCL10.
In conclusion, IKKis targeting TBK1 and IKKε present significant HIV-1 reactivation capacity in lymphoid and myeloid cellular models. The low toxicity in the absence of cell activation suggests their use as alternative LRAs targeting both lymphoid and myeloid compartments avoiding hyperinflammatory responses in chronic HIV-1 infection.

Cells
PBMCs were obtained from the blood of healthy donors using a Ficoll-Paque density gradient centrifugation and CD4+ T lymphocytes were purified using negative selection antibody cocktails (StemCell Technologies, Vancouver, BC, Canada) as described previously [34]. Buffy coats were purchased from the Catalan Banc de Sang i Teixits. The buffy coats received were totally anonymous and untraceable and the only information given was whether or not they have been tested for disease. PBMCs or CD4+ T lymphocytes were kept in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Billings, MT, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin with IL-2 (16 U/mL) and then treated with the corresponding compounds for 48-72 h.
HIV+ individuals: All participants in the study provided informed consent and samples were processed as mentioned above. Subject samples were included if the individuals were older than 18 years old, had chronic HIV-1 infection, and had previously been on highly active ART for >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 entry. The immunological and virological characteristics from all participants are found in supplementary Table S2.

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.

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% CO 2 . 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.
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 a whole in each treatment condition using the optimized t-distributed stochastic neighbor embedding (opt-SNE) algorithm for dimensional reduction and visualization.

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 Scientific) 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].

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.

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.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.