The effect of ARVs on the MEKKK1 gene promoter, inflammatory cytokine expression and signalling in acute treated Jurkat T cells

Abstract ARVs alter the methylation status of the MEKKK1 gene promoter in acute treated Jurkat T cells with inflammatory outcomes Inflammation is reduced in patients under going antiretroviral therapy; however the mechanism is not well understood. We investigated DNA methylation of the mitogen-activated protein kinase kinase kinase kinase 1 (MEKKK1) gene promoter in Jurkat T cells to determine whether the antiretroviral drugs, lamivudine, tenofovir disoproxil fumarate, dolutegravir, TLD (a combination of TDF, 3TC and DTG) and efavirenz modify the methylation status of the MEKKK1 gene – a known stimulus of inflammation. Acute antiretroviral treatments (24 h) were not cytotoxic to Jurkat T cells. MEKKK1 promoter hypomethylation occurred in cells treated with 5-aza-2’-deoxycytidine (Aza), TDF and 3TC, and MEKKK1 promoter hypermethylation occurred in cells treated with DTG; however, promoter DNA methylation of the MEKKK1 gene did not influence MEKKK1 gene expression; therefore, these drugs did not epigenetically regulate MEKKK1 and downstream signalling by promoter DNA methylation. Acute TLD and EFV treatments induced inflammation in Jurkat T cells by increasing MEKKK1, MAPK/ERK and NFκB expression, and activating tumour necrosis factor-α (TNF-α) expression. ARVs decreased IL-10 gene expression, showing no anti-inflammatory activity. The data shows that the inflammation caused by ARVs is not related to the methylation status of MEKKK1 gene promoter and suggests an alternative stimulus via post-transcriptional/post-translational modifications may activate the canonical MEKKK1/NFκB pathway that leads to inflammation. Finally, an increase in NFκB activity and pro-inflammatory cytokine activation seemed to occur via the MAPK/ERK pathway following ARV treatments in Jurkat T cells.


Introduction
Antiretroviral therapy (ART) is a life-long treatment that increases the life expectancy of patients; therefore, it is necessary to study the effects of prolonged ART usage (Hattab et al. 2014;Wandeler et al. 2016). ART has been shown to reduce inflammation levels and its contributing mediators; however, the impact of different antiretroviral drugs (ARVs) from different classes on inflammation remains unclear (Lederman et al. 2013;Hattab et al. 2014;Hileman and Funderburg 2017). Studies investigating the effects of first-line ART (which include ARVs such as tenofovir disoproxil fumarate (TDF), lamivudine (3TC) and efavirenz (EFV)) on the biomarkers of inflammation have produced conflicting results (Hattab et al. 2014;Sandler and Sereti 2014). In addition, studies involving combined ARV treatment strategies to further reduce inflammation compared to that of single-dose ARVs have not produced consistent results (Sandler and Sereti 2014). Thus, there is a need for controlled studies which compare inflammatory biomarkers following different ART treatments to prepare for clinical inflammatory events.
Inflammation is a normal process if regulated; however dysregulation can result in permanent tissue damage and lead to acute and chronic inflammation (Liu et al. 2017). Inflammation occurs in response to a stimulus, causing swelling, heat, pain and redness, eventually leading to the recruitment of immune cells, and the release of additional inflammatory mediators (Khalaf et al. 2010;Sandler and Sereti 2014). Cytokines, primarily produced by T-lymphocytes (T-cells) and macrophages, make up one class of inflammatory biomarkers and are the chief mediators of inflammatory responses, with either pro-inflammatory or anti-inflammatory abilities (Brenner et al. 2014). Elevated levels of pro-inflammatory cytokines, for example, tumour necrosis factor (TNF) and interleukin-6 (IL-6) have been linked to diseases, which include pulmonary and cystic fibrosis, and may be elevated in AIDS (Zhang and An 2007;Khalaf et al. 2010).
Pro-inflammatory cytokine gene promoter activity is directed by the transcription factor, NFjB (nuclear factor kappa-light-chain-enhancer of activated B-cells), which has been shown to induce (interleukin) IL-6 gene activity through tumour necrosis factor-a (TNF-a) (Luo and Zheng 2016). Research shows that members from the TNF receptor superfamily are well-described inducers of NFjB (Hayden and Ghosh 2014;Liu et al. 2017). Once NFjB is activated and translocates to the nucleus, various pro-inflammatory genes are transcribed, including IL-6 (Dhingra et al. 2009;Liu et al. 2017). The anti-inflammatory cytokine, interleukin-10 (IL-10) is a known inhibitor of pro-inflammatory cytokines and achieves this by suppressing NFjB activation and preserving IjB (nuclear factor of kappa light polypeptide gene enhancer in B-cells) protein expression (Dhingra et al. 2009;Iyer and Cheng 2012;Su et al. 2012).
Studies show that mitogen-activated protein kinase kinase kinase kinase 1 (MEKKK1) is a novel functional component of the IjB kinase (IKK) complex that is important for T-cell receptor (TCR) mediated NFjB activation (Brenner et al. 2005). MEKKK1, also known as haematopoietic progenitor kinase 1 (HPK1) or MAP4K1, is comprised of a kinase domain located on an N-terminus and a regulatory domain located on the C-terminus, referred to as the citron homology domain . In epithelial cells, for example, when full-length MEKKK1 is activated through ectopic expression it selectively activates the stress-activated protein kinases/Jun amino-terminal kinases (SAPK/JNK) as well as the NFjB pathways (Brenner et al. 2005). Once MEKKK1 activates the IKK complex via TCR stimulation, phosphorylation targets IkB, resulting in the degradation of the IkB by the 26S proteasome, allowing NFjB proteins to be released into the nucleus, followed by gene transcription and NFjB pathway activation ( Figure 1) (Moses et al. 2021). DNA methylation is an epigenetic process that is carried out through the chemical change of cytosine by covalently adding a methyl group to its 5-carbon in a way that influences cell signalling and regulates gene expression (Abdel-Hameed et al. 2016). DNA methylation occurs at cytosines located 5 0 of guanines or CpG dinucleotides and studies show that unmethylated CpG dinucleotides called CpG islands are located in the promoter and first exon regions of $60% of all genes (Huang et al. 1999). At present, we know that chromosome 19 of the MEKKK1 gene has been mapped and shows a transcript length of 2,700 base pairs, translation length of 833 residues, 32 exons and 32 coding exons, and highlights the promoter regions of the MEKKK1 gene ( Supplementary Figures 1 and  2) (Ensembl GRCh37). Studies looking at the epigenetics of the MEKKK1 gene are limited and while they explain the DNA methylation process and its involvement in gene expression, these studies have not epigenetically mapped out the MEKKK1 gene promoter, showing the coordinates, length and density of CpG islands (Altorok and Sawalha 2013;Long et al. 2016;Farivar and Aghamaleki 2018). While gene promoter hypermethylation inhibits gene transcription and stops the binding of transcription factors, gene promoter hypomethylation activates gene transcription (Moses et al. 2021). Furthermore, since studies show that MEKKK1 activates the NFjB pathway in Jurkat T cells and that MEKKK1-mediated NFjB activation is independent of MEKKK1-mediated SAPK/JNK activation  this prompts further investigation into the MEKKK1/NFjB pathway. Due to the increase in MEKKK1 gene expression levels leading to IKK activation, IkB degradation, and subsequent NFjB activation, it is possible that ARVs could epigenetically modify the methylation status of the MEKKK1 gene promoter to decrease the transcription of NFjB, curtail pro-inflammatory gene transcription and reduce inflammation.
To determine whether ARVs affect the MEKKK1 gene promoter, the methylation status of the MEKKK1 promoter region was analysed in Jurkat T cells treated with TDF, 3TC, DTG, TLD (a combination of TDF, 3TC and DTG) and EFV. Since studies show that ARVs reduce inflammation, our aim is to determine the effect of acute treatments of these ARVs on the MEKKK1/NFjB inflammatory pathway by monitoring MEKKK1, NFjB and MAPK/ERK expression, as well as proinflammatory cytokine (TNF-a and IL-6) and anti-inflammatory (IL-10) cytokine expression.

Materials
DNA methylation inhibitor, 5-aza-2 0 -deoxycytidine (Aza; A3653), was obtained from Sigma-Aldrich. Primer sequences for qPCR and DNA methylation studies were purchased from Inqaba Biotechnical Industries (Pty Ltd), South Africa. The forward (TTGTAGGGATGGGTTCTTGC) and reverse (ATCTTGGGGACTGCAAATGA) sequences for the MEKKK1 gene promoter were found in published research (Zhang et al. 2011) before being purchased from Inqaba Biotechnical Industries (Pty Ltd), South Africa. The Jurkat T cell line was donated by the HIV Pathogenesis Programme laboratory at the University of KwaZulu-Natal, South Africa. The OneStep qMethyl TM kit (ZR D5310) was obtained from Inqaba Biotechnical Industries (Pty) Ltd. Consumables used in tissue culture were purchased from Lonza Biotechnology (Basel, Switzerland). All other reagents used were obtained from Merck (Darmstadt, Germany). Antiretroviralstenofovir (TDF), lamivudine (3TC), dolutegravir (DTG) and efavirenz (EFV) were donated by Dr Sooraj Baijnath (National Institutes of Health, South Africa).

Cell culture and treatment
Jurkat T cells (1 Â 10 6 cells/ml) were grown (37 C, 5% CO 2 ) in RPMI-1640 containing 20% foetal calf serum). A haemocytometer was used to count the cells once they reached confluency and the equation (C 1 V 1 ¼ C 2 V 2 ) was used to ensure that 1 Â 10 6 cells/ml was used in all experiments. Mean steady-state peak plasma concentration (C max ) is considered to be the most physiologically relevant ARV treatment concentration that represents naturally occurring drug concentrations following their intake (Thabethe et al. 2015). Therefore, we used C max concentrations in the treatment of Jurkat T cells for 24 h. The C max concentrations of the ARVs used in this study are as follows: TDF (0.3 mg/ml) (Thabethe et al. 2015), 3TC (1.5 mg/ml) (Reddy and Mohanambal 2011), DTG (3.67 mg/ml) (Ballantyne and Perry 2013) and EFV (4.07 mg/ml) (Thabethe et al. 2015). DMSO (4.07 mg/ml) was included as a vehicle control. ARVs were first dissolved in 5 ml sterile dH 2 O before being diluted in RPMI-1640 to obtain the desired C max . The cells underwent incubation for 24 h with and without treatments (37 C, 5% CO 2 ). An untreated control (cells and culture media) was prepared. The Aza (50 mM) stock solution was made up in DMSO (100%). A 10 lM (24 hr) treatment of Aza (Aza 10) was obtained from literature and used as a negative control, to induce DNA hypomethylation in the cells (Ghazi et al. 2019). The Trypan blue cell exclusion method was performed to determine cell viability. All experiments were performed in triplicate and two independent times.

ATP assay
The ATP CellTitre Glo reagent (Promega, Madison, USA) was utilised to quantify ATP and determine cell viability in Jurkat T cells. Jurkat T cells (20000 cells/well), following ARV treatment, were seeded in triplicate onto an opaque polystyrene 96-well microtitre plate. 20 ll of ATP-Glo reagent was placed into each well and samples were incubated for 30 min in the dark (RT). Luminescence was determined using the Modulus TM microplate luminometer and data was expressed as RLU.

DNA isolation and promoter methylation of MEKKK1
DNA was isolated from the controls, Aza and ARV-treated Jurkat T cells. Cells were incubated in 600 ml of cell lysis buffer (0.5 M EDTA (pH 8.0), 1 M Tris-HCl (pH 7.6), 0.1% SDS) and back-pipetted every 5 min using an insulin syringe for 15 min (RT). Thereafter, 600 ll of potassium acetate buffer (5 M potassium acetate in glacial acetic acid) was added and samples were manually inverted for 8 min (RT); before centrifugation (13,000 Â g, 5 min, 24 C). The supernatants, containing genomic DNA, were transferred into 1.5 ml micro-centrifuge tubes, treated with 600 ll of 100% isopropanol and then centrifuged at 13000 Â g for 5 min at 24 C. The samples were then washed with 300 ll of 100% ethanol and centrifuged (13000 Â g, 5 min, 24 C). The samples were air dried for 30 min (RT), resuspended in 40 ml of DNA hydration buffer (RT, 10 mM EDTA (pH 8.0), 100 mM Tris-HCl (pH 7.4) and heated (65 C, 15 min). Isolated DNA was then purified with the DNA Clean and Concentrator TM -5 Kit (Zymo Research, D4003), according to manufacturer's instructions. The Nanodrop2000 spectrophotometer (Thermo-Fischer Scientific) was used to determine DNA concentration, which was then standardised to 4 ng/ml. DNA purity was assessed using the A260/A280 absorbance ratios. The OneStep qMethyl Kit (Zymo Research, 5310) was used to analyse the promoter methylation of MEKKK1, as per manufacturer's instructions. DNA (20 ng) was added to test and reference reaction mixtures containing HPK1 promoter F and HPK1 promoter R primers. Cycling conditions involved the following: digestion by methyl sensitive restriction enzymes (AccII, HpaII, and HpyCH4IV) (37 C, 2 h), initial denaturation (95 C, 10 min), followed by 45 cycles of denaturation (95 C, 30 s), annealing 58 C, extension (72 C, 60 s), final extension (72 C, 60 s), and a hold at 4 C. The percentage methylation was calculated using the 2 ÀDDCt method (Livak and Schmittgen 2001) and all data were presented as fold-changes relative to the control.

RNA isolation and quantitative polymerase chain reaction (qPCR)
RNA was extracted from control, Aza-treated Jurkat T cells and ARV treated Jurkat T cells using Qiazol reagent (Qiagen, 79306). Cells were washed with 0.1 M PBS followed by 5 min (RT) incubation in 500 ll Qiazol and 500 ll 0.1 M PBS. Cell lysates were allowed to incubate overnight (À80 C). Following the addition of chloroform (100 ll), the samples were centrifuged (12000 Â g, 15 min, 4 C. The RNA-containing supernatant was placed into 1.5 ml micro-centrifuge tubes, thereafter 250 ll of 100% cold isopropanol was added to each sample and left to incubate overnight (À80 C). Samples were then centrifuged (12000 Â g, 20 min, 4 C) and the pellets containing RNA were then washed with 500 ll of 75% cold ethanol. Samples were centrifuged (7400 Â g, 15 min, 4 C), followed by air drying for 30 min (RT), re-suspension in 15 ll of nuclease-free water and incubation for 3 min (RT). The Nanodrop2000 spectrophotometer was used to quantify RNA, which was then standardised to 1000 ng/ll. The purity of RNA was assessed using the A260/A280 absorbance ratios. The Maxima H Minus First Strand cDNA Synthesis Kit (Thermo-Fischer Scientific, K1652) was then used to prepare cDNA, as per the manufacturer's instructions. Thermocycler conditions were 25 C for 5 min, 42 C for 45 min, 85 C for 5 min and a final hold at 4 C. The PowerUp SYBR Green Master Mix [ThermoFisher Scientific, catalogue number: 25742], was used to determine MEKKK1, NFjB, IL-6, IL-10 and TNFa mRNA levels, according to instructions provided by the manufacturers. mRNA expressions were normalised using the GAPDH housekeeping gene. The CFX96 Real Time PCR System (Bio-Rad) was used to perform qPCR experiments and analyses were done using the Bio-Rad CFX Manager TM . CFX96 Thermocycler conditions were as follows: initial denaturation (8 min, 95 C), followed by 40 cycles of denaturation (95 C, 15 s), annealing (Table 1, 40 s) and extension (72 C, 30 s). The Bio-Rad CFX Manager TM Software version 3.1 was used to analyse all data and relative changes in expression was determined using the comparative threshold cycle (Ct) method (Ghazi et al. 2019).

Western blots
Protein expression of MEKKK1, MAPK1, NFjB, IL-6 and TNFa were determined using Western blots. All experiments were run in triplicate. Following a 24 h incubation in 25 cm 3 cell culture flasks, the controls, and ARV treated cells were rinsed with 0.1 M PBS for three times. Cytobuster TM Reagent (200 ml) (Novagen, San Diego, CA, catalogue no. 71009) was used to lyse the cells, whereby, the cells were incubated for 30 min on ice before mechanical lysing using an insulin syringe. Cell lysates were then placed into 1.5 ml micro-centrifuge tubes and centrifuged (10000 Â g, 10 min, 4 C). The crude protein extract, contained in the supernatant, were then placed into 1.5 ml micro-centrifuge tubes and stored at À80 C overnight. Crude protein was then quantified using the Bicinchoninic Acid (BCA) Assay and the protein samples were standardised to a 1.5 mg/ml concentration. Following preparation in Laemmli buffer [dH 2 O, 0.5 M Tris-HCl (pH 6.8), glycerol, 10% SDS, 5% beta-mercaptoethanol, and 1% bromophenol blue] the standardised samples were boiled for 5 min at 100 C.
The Bio-Rad compact power supply was used to perform electrophoresis (1 hr, 150 V) on the protein samples and the molecular weight marker in sodium dodecyl sulphate polyacrylamide gels, and thereafter electro-transferred to a nitrocellulose membrane using the Bio-Rad Transblot Turbo Transfer System. These membranes were then blocked with 1:5000 5% bovine serum albumin (BSA) (phosphorylated proteins) in Tris-buffered saline containing 0.05% Tween 20 [TTBS; 150 mM NaCl, 3 mM KCl, 25 mM Tris, 0.05% Tween 20, pH 7.5] for 1 hr (RT) with gentle shaking. The membranes were probed with primary antibodies -HPK1 (ThermoFisher -PA575258), MAPK1 (91025), NFjB (8242S), IL-6 (12153S) and TNFa (37075) all obtained from Cell Signalling Technology; 1:5000 in 5% BSA for 1 hr (RT). Membranes were then washed five times with TTBS (10 min per wash) and incubated with rabbit (Cell Signalling -7074S) secondary antibody 1:10,000 for 1 hr (RT) on a shaker. The membranes were washed for five times in TTBS (10 min per wash at RT) and the Clarity TM Western ECL Substrate Kit (#170-5060, Bio-Rad) was used to visualise protein bands, which were detected for 30 min using the ChemiDoc TM XRS Molecular Imaging System (Bio-Rad). Results were displayed as relative band density (RBD) compared to the control. Following detection, 5% of hydrogen peroxide was used to strip the membranes for 30 mins at 37 C, which were then washed three times with TTBS for 10 min (RT), subsequently blocked for 1 hr (RT) in 5% BSA made up in TTBS and probed for 30 min (RT) with the house keeping protein antibody, b-actin (Sigma Aldrich -a3854) which was made up as 1:5000 dilutions in 5% BSA. b-actin was used to normalise the proteins of interest.

Statistical analysis
Statistical analyses were performed using the statistical analysis software, GraphPad Prism v5.0 (GraphPad Software Inc., La Jolla, USA). The one-way ANOVA with the Bonferroni multiple comparisons test and the unpaired T-test with Welch's correction were used to assess the differences between samples, with p < 0.05 as the level of significance (p).

ARVs increase metabolic activity and cell viability in jurkat T cells
To determine whether acute treatment of Jurkat T cells with ARVs for 24 h was cytotoxic, the WST-1 cell proliferation/viability assay was carried out (Figure 2(A)). Compared to the control, TDF, 3TC, DTG, TLD and EFV treatments did not inhibit the ability of Jurkat T cells to cleave the WST-1 reagent/tetrazolium salt via NADH-dependent mitochondrial dehydrogenases. Hence, acute ARV treatments significantly increased the proliferation/metabolic activity of Jurkat T cells.
In addition to the WST-1 assay, the adenosine triphosphate (ATP) assay was done to evaluate cell viability ( Figure  2(B)). Compared to the control, Jurkat T cells treated with TDF, 3TC, DTG, TLD and EFV for 24 h showed an increase in ATP content. 3TC significantly increased ATP activity in Jurkat T cells (p ¼ 0.01). Hence, acute ARV treatments did not reduce cell viability in Jurkat T cells. Table 1. Annealing temperatures of primer sequences used in qPCR assays.

Gene
Forward sequence Reverse sequence Annealing Temperature ( C)  (Figure 4(A)). TDF, 3TC, DTG and EFV did not influence MEKKK1 gene expression in Jurkat T cells (Figure 4(A)). TLD (p ¼ 0.02) significantly increased MEKKK1 gene expression in Jurkat T cells (Figure 4(A)). MEKKK1 protein expression decreased significantly following ARV treatments compared to the control in Jurkat T cells (Figure 4(B)). The transcription factor, NFjB is upregulated during inflammation and is considered a chief regulator of pro-inflammatory cytokines. Following IKK complex activation and IkB degradation by MEKKK1, NFjB proteins are released into the nucleus, followed by gene transcription and NFjB pathway activation. NFjB gene expression increased significantly following TLD (p ¼ 0.04) and EFV (p ¼ 0.02) treatments in Jurkat T cells ( Figure  4(C)). NFjB protein expression significantly decreased following 3TC (p ¼ 0.0003) treatment and increased significantly following DTG (p ¼ 0.0002), TLD (p ¼ 0.0001) and EFV (p ¼ 0.02) treatments in Jurkat T cells (Figure 4(D)). MAPK signalling stimulates and transcriptionally regulates NFjB, and ERK1/2 activates canonical NFjB signalling which has been shown to regulate pro-inflammatory gene expression (Nakano et al. 2020). p 44 MAPK/ERK1 protein expression significantly increased following acute ARV treatments in comparison to the control in Jurkat T cells (Figure 4(E)). p 42 MAPK/ERK2 protein expression significantly increased following ARV treatments except TDF (p ¼ 0.001), which significantly decreased p 42 MAPK/ERK2 protein expression in Jurkat T cells (Figure 4(E)).
Effect of ARVs on TNF-a, IL-6 and IL-10 gene and protein expressions Once NFjB is activated and translocates to the nucleus, various pro-inflammatory genes are transcribed. The pro-inflammatory  cytokine, tumour necrosis factor-alpha (TNF-a) has been shown to play a role in activating intracellular signalling of NFjB and MAPK (Mitoma et al. 2018). TNF-a gene expression significantly increased following TLD (p ¼ 0.0002) and EFV (p ¼ 0.02) treatments in Jurkat T cells (Figure 5(A)). TDF, 3TC and DTG did not influence TNF-a gene expression compared to the control in Jurkat T cells. TNF-a protein expression significantly decreased compared to the control following ARV treatments in Jurkat T cells (Figure 5(B)). IL-6 gene expression decreased significantly following ARV treatments compared to the control in Jurkat T cells ( Figure 5(C)). IL-6 protein expressions increased significantly following ARV treatments, except TDF (p ¼ 0.004) and 3TC (p ¼ 0.0003) which decreased IL-6 protein expression significantly in Jurkat T cells (Figure 5(D)).
IL-10 is an anti-inflammatory cytokine that is induced by TNF-a following inflammation or a stress reaction (Stenvinkel et al. 2005). Following ARV treatments, anti-inflammatory activity was assessed in Jurkat T cells by measuring the gene expression of IL-10, which mediates anti-inflammatory responses and the expression of pro-inflammatory cytokines through MAPK/NFjB activities. Using qPCR, IL-10 gene expression significantly decreased following ARV treatments compared to the control in Jurkat T cells (Figure 5(E)).

Discussion
ARVs have been shown to reduce inflammation; however the exact pathway of this interaction has not been well documented. Furthermore, the effect of ARVs on epigenetic modifications, such as DNA methylation on the MEKKK1 gene, a known stimulus of the NFjB inflammatory pathway is not well documented. Since DNA methylation is known to influence cell signalling and regulate gene expression we aim to determine the effect of ARVs on the methylation status of the MEKKK1 gene promoter and MEKKK1 gene expression in Jurkat T cells. The MEKKK1 gene is a novel functional component of the IKK complex that is important for TCR mediated NFjB activation and inflammation is largely driven by proinflammatory cytokines which are regulated by NFjB, such as interleukin-6 (IL-6) and TNF-a (Acchioni et al. 2019;Moses et al. 2021). In this study, we provide evidence on the methylation status of the promoter region of the MEKKK1 gene in response to ARV treatment, with tenofovir disoproxil fumarate (TDF), lamivudine (3TC), dolutegravir (DTG), efavirenz (EFV) and a combination drug made up of TDF, 3TC and DTG called TLD which has not been documented to our knowledge.
Inflammation can lead to hyperproliferation and DNA damage in cells (Kiraly et al. 2015). The investigation of cell metabolic activity and cell viability is fundamental to evaluate the biological reaction of cells to outside stimuli (Yin et al. 2013). Studies evaluating metabolic activity and cell viability in Jurkat T cells following ARV treatments are limited. The WST-1 assay showed that acute ARV treatment of TDF, 3TC, DTG, EFV and TLD over 24 h increased metabolic activity; this indicated that acute ARV treatments did not hinder mitochondrial function in Jurkat T cells (Figure 1(A)). This is in agreement with previous studies which showed that TDF, 3TC, DTG and EFV do not hinder cell metabolic activity via the WST-1 assay in vitro (Zhang et al. 2015;Argaw et al. 2016). Furthermore, the exposure of cells to injurious agents can lead to cell membrane and DNA damage, pathway disruption, ATP depletion and mitochondrial damage, which further damages nucleic acids and proteins (Miller and Zachary 2017). While acute treatments of TDF, DTG, TLD and EFV over 24 h in Jurkat T cells did not decrease ATP activity, 3TC significantly increased ATP activity (Figure 1(B)); this indicated that acute ARV treatments increased cell viability in Jurkat T cells. This is in agreement with previous studies which showed that metabolic reactions form 3TC-triphosphates which increase intracellular ATP concentrations (Garc ıa-Trejo et al. 2021). Hence, cell viability results show that TDF, 3TC, DTG, TLD and EFV are not cytotoxic; which is supported by previous studies (Moraes Filho et al. 2016;Mendelsohn and Ritchwood 2020;George et al. 2021).
Chronic inflammation has been shown to induce global DNA hypomethylation (Niwa and Ushijima 2010). However, the target genes for DNA methylation induced by chronic inflammation have been poorly addressed and studies suggest that the promoter regions of these target genes should be focussed on. The MEKKK1 gene is a known activator of the NFjB-mediated inflammatory pathway; hence, we examined inflammatory response in relation to MEKKK1 gene promoter methylation and MEKKK1 gene expression in Jurkat T cells treated with ARVs. Tissue-or cell-specific DNA hypomethylation or hypermethylation influence gene expression at promoter regions that are rich in CpGs (Ehrlich 2019). DNA hypomethylation is associated with an increase in gene expression and DNA hypermethylation plays a role in silencing gene promoters due to extensive DNA methylation near the transcription start site (Ehrlich 2019). Aza significantly increased MEKKK1 gene expression in Jurkat T cells ( Figure  4(A)); this correlated with MEKKK1 gene promoter hypomethylation results, indicating that Aza induced global hypomethylation in Jurkat T cells (Figure 3). TDF and 3TC induced significant MEKKK1 gene promoter hypomethylation in Jurkat T cells ( Figure 2); this indicated that these ARVs altered the methylation status of the MEKKK1 gene promoter by decreasing the content of 5-methylcytosine in the MEKKK1 genome. DTG induced significant hypermethylation of the MEKKK1 gene promoter (Figure 2); this is due to an increase in methyl groups added to the MEKKK1 gene promoter region by DTG, which has electron-donating methyl groups in its structure (Schreiner et al. 2016).
Although TDF and 3TC hypomethylated and DTG hypermethylated MEKKK1 promoter DNA; TDF, 3TC and DTG did not influence MEKKK1 gene expression (Figure 4(A)). Since there is no correlation between MEKKK1 promoter methylation and MEKKK1 gene expression patterns, we report that these drugs do not regulate MEKKK1 expression by promoter DNA methylation. Furthermore, MEKKK1 protein expression decreased significantly following ARV treatments compared to the control in Jurkat T cells (Figure 4(B)). This did not correlate with the increase in MEKKK1 gene expression in TLD treated Jurkat T cells (Figure 4(A)); suggesting that acute TLD treatments induced post-translational modifications of MEKKK1 proteins in Jurkat T cells. Post-translational modifications alter the properties of proteins by proteolytic cleavage and the addition of modifying groups, such as methyl, phosphoryl, acetyl and glycosyl to one or more amino acids in a way that affects the dynamic and structure of proteins. These modifications alter gene expression regulation and activation. Hence, it is possible that the acute ARV treatments in this study may have contributed modifying groups from their structures which could have affected MEKKK1 protein expression.
Once MEKKK1 is activated, it activates the NFjB pathway via TCR stimulation, allowing NFjB proteins to be released into the nucleus, followed by NFjB gene transcription (Moses et al. 2021). The increase in MEKKK1 gene expression in TLD treated Jurkat T cells correlated with an increase in NFjB gene (Figure 3(C)) and NFjB protein (Figure 3(D)) expressions; this indicated that TLD increased MEKKK1 expression to activate the NFjB pathway. Although EFV significantly increased NFjB gene (Figure 3(C)) and NFjB protein (Figure 3(D)) expressions, EFV did not significantly increase MEKKK1 gene and protein expressions; this indicated that another pathway was involved in NFjB activation. Extracellular signal-regulated kinase (ERK), belonging to the mitogen-activated protein kinase (MAPK) family, mainly occurs in the forms ERK1 (p44) and ERK2 (p42) and is known to play a role in inflammation via the ERK1/2 pathway by increasing NFjB activity (Lu and Malemud 2019). ARVs increased MAPK/ERK1/2 protein expression in Jurkat T cells (Figure 3(E)); hence, we suggest that EFV activated the MAPK/ERK pathway, which lead to NFjB activation in Jurkat T cells. This is in agreement with previous studies which show that acute treatment of EFV triggers inflammation via NFjB in vitro by increasing the phosphorylation of MAPK/JNK and IjBa, to increase NFjB translocation to the nucleus (Jamaluddin et al. 2010;Alegre et al. 2022).
Once NFjB is activated and translocates to the nucleus, various pro-inflammatory genes are transcribed. Inflammation regulation via the canonical NFjB pathway occurs in response to stimuli, for example, cytokine receptor ligands, members of the TNF receptor family and T-cell receptors (Liu et al. 2017). The NFjB binding site is also crucial for the IL-6 promoter to respond to TNF-a (Luo and Zheng 2016). The increase in TNF-a gene expression in TLD and EFV treated Jurkat T cells ( Figure  4(A)) correlated with an increase in MEKKK1 gene expression and NFjB gene, and protein expression; this indicated that TLD and EFV activate MEKKK1 and MAPK respectively to mediate the NFjB inflammatory pathway in Jurkat T cells. Previous studies show that when TNF-a is increased, phospho-IjBa levels become elevated which increases NFjB translocation to the nucleus (Jamaluddin et al. 2010). Although TLD and EFV increased TNF-a gene expression, ARVs significantly decreased TNF-a protein expression in Jurkat T cells (Figure 4(B)); this indicated that TLD and EFV induced post-translational modifications of the TNF-a gene. TDF and 3TC decreased IL-6 gene expression in Jurkat T cells (Figure 4(C)), which correlated with a decrease in IL-6 protein expression (Figure 4(D)); this indicated that TDF and 3TC do not activate the NFjB inflammatory pathway to stimulate the release of IL-6. This is in agreement with studies showing that TDF and 3TC do not increase IL-6 inflammatory markers in patients receiving treatment (Funderburg et al. 2016;Calza et al. 2022). Although acute treatments of DTG, TLD and EFV increased IL-6 protein in Jurkat T cells (Figure 4(D)), this did not correlate with a decrease in IL-6 gene expression; this indicated that DTG, TLD and EFV induced post-translational modifications of the IL-6 gene. MicroRNAs (MiRNAs) are known regulators of post-transcriptional modifications in gene expression. MiRNAs target specific mRNA sequences for degradation or to inhibit translation and are capable of repressing DNA methylation (Ghazi et al. 2019). Previous studies show that acute EFV treatment increases IL-6 gene expression in healthy cells in vitro; however studies also show that chronic EFV treatment decreases IL-6 levels in HIV-infected CD4 þ T cells. Furthermore, it has been shown that a combination treatment of DTG/3TC decreases pro-inflammatory IL-6 markers in patients (Calza et al. 2022).
Chronic inflammation is made worse in the absence of the anti-inflammatory cytokine, IL-10. IL-10 blocks pro-inflammatory gene expression by blocking transcription factors or inhibiting the expression of genes (Murray 2006;Iyer and Cheng 2012;Islam et al. 2021). TNF-a has been shown to stimulate the release of IL-10 from various cells (Stylianou et al. 1999). Acute ARV treatments decreased IL-10 gene expression in Jurkat T cells (Figure 4(E)); this indicated that acute ARV treatments did not activate anti-inflammatory response in Jurkat T cells. The results show that ARVs caused post-transcriptional/post-translational changes to decrease TNF-a gene and/or protein expression ( Figure 5(B)); hence, ARVs may have affected TNFa-mediated IL-10 release in Jurkat T cells.

Conclusion
This study showed that although TDF, 3TC, DTG, EFV and TLD are not cytotoxic to Jurkat T cells; they may induce inflammation by influencing pro-inflammatory cytokine expression and signalling. The results indicated that TDF, 3TC and DTG alter the methylation status of the MEKKK1 gene promoter, with TDF and 3TC inducing MEKKK1 promoter hypomethylation, and DTG inducing MEKKK1 promoter hypermethylation in Jurkat T cells. However, promoter DNA methylation of the MEKKK1 gene did not influence MEKKK1 gene expression; therefore, these drugs did not epigenetically regulate MEKKK1 by promoter DNA methylation. The results further show that TLD and EFV increase MEKKK1, NFjB, MAPK/ERK and TNF-a expression; indicating that these drugs may activate the NFjB inflammatory pathway and stimulate the expression of pro-inflammatory cytokines. ARVs did not influence IL-6 activity in Jurkat T cells and decreased IL-10 gene expression, showing that these drugs affect the anti-inflammatory pathway in Jurkat T cells. Overall, these results show that TLD and EFV may contribute to chronic inflammation over long-term usage; however, this study did not find any evidence that the influence of ARVs on MEKKK1, NFjB, MAPK/ERK, TNF-a, IL-6 and IL-10 expressions are epigenetically regulated via promoter DNA methylation of MEKKK1 gene. Future studies should focus on silencing DNA methyltrasferases in cells treated with these ARVs and look at downstream cytokines and signalling to determine whether these drugs alter the methylation status of the MEKKK1 gene promoter to cause inflammation. We suggest that post-transcriptional and post-translational studies should be carried out since results indicate that these drugs may induce post-transcriptional and post-translational modifications of genes and proteins involved in the NFjB inflammatory pathway. We recommend that since NF-jB regulates inflammatory factors through phosphorylation, future studies should look at the detection of phosphorylated NF-jB protein. In vivo studies are also necessary to implement these findings into patient care. Since this study was limited to a transformed cell line, future studies should be carried out on primary cell cultures that are isolated from parental tissue as these models better represent the in vivo situation.