Immune response of human cultured cells towards macrocyclic Fe₂PO and Fe₂PC bioactive cyclophane complexes

Cell culture and experimental dosing

Human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 8% fetal bovine serum (FBS). The media was supplemented with an antibiotic cocktail (penicillin, streptomycin, and amphotericin). HEK cells were grown to 80% confluence in 6-well plates at 37 °C with humidity and 5% CO₂, and then treated with an aqueous solution of Fe₂PO or Fe₂PC, to a final concentration of 120 µM. Sterile milliQ water was used as a negative control. Synthesis and characterization of the Fe₂PO and Fe₂PC cyclophanes has been previously described (Salazar-Medina et al., 2013). Each experiment was performed in duplicate.

Gene expression analysis

Cells were harvested from each cyclophane complex treatment at time points of 8 and 24 h. The RNeasy Mini Kit (Qiagen-SABiosciences, Frederick, MD, USA), which included a step to remove genomic DNA, and the RT² First Strand Kit (Qiagen-SABiosciences, Frederick, MD, USA) was used for synthesizing cDNA by reverse transcription. The RT² Profiler™ PCR Array Human Innate and Adaptive Immune Responses, specific for 84 immune genes (catalog PAHS-052Z; Qiagen-SABiosciences, Frederick, MD, USA) was used to query immune responsive gene expression. This method consists of a quantitative PCR reaction with SYBR-green detection, where the cDNA is loaded into 96-well PCR array plates. Each plate has specific primers for the 84 immune genes, plus housekeeping genes and experimental controls. The protocol and recommendations of the manufacturer were followed thoroughly.

The experiment was performed on a Roche LightCycler 480 (Roche, Indianapolis, IN, USA) thermal cycler and the amplification conditions were: one cycle of 95 °C for 10 min, then 45 cycles of 95 °C for 15 s, and 60 °C for 1 min. Raw data were acquired and analyzed using the Roche LightCycler 480 software, including the calculation of C_t (threshold cycle) values, which are proportional to the abundance of the cDNA of interest (Romoser et al., 2011).

Calculation of gene expression changes

The RT² Profiler™ PCR Array Human Innate and Adaptive Immune Responses array includes five housekeeping genes: β₂-microglobulin, hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a, glyceraldehyde-3-phosphate dehydrogenase (GADPH), and β-actin. Among those, the β₂-microglobulin gene (B2M) expression showed less variance across the sample conditions. Therefore, B2M was utilized for subsequent normalization. The 2^−ΔΔCt method was used to estimate changes in relative gene expression (Livak & Schmittgen, 2001), and the fold changes were calculated for the expression level of the genes in the respective control group. For instance, a 2.0-fold change of gene A indicates that the expression of gene A was twice as abundant in the samples treated with the cyclophane compared to the expression in the control group. Meanwhile, 0.5-fold change of gene B suggests that the expression of this gene was two times less in the cyclophane group compared to its expression in the untreated group (Romoser et al., 2011).

Changes in gene expression

HEK cells were used to determine gene expression changes after treatment with Fe₂PO and Fe₂PC using an exposure concentration of 120 µM and two time points (8 or 24 h). Genes assayed in this study can contribute to four physiological response categories (inflammation, apoptosis, oxidative stress, and non-inflammatory immune responses) as shown in Table 1. Many of the genes have functions in more than one of these interdigitating pathways. According to this classification, specific results are discussed below, in the order of decreasing observed toxicological responses.

The results for the 29 genes with complete data for all samples are presented in a heat map (Table 2). Fold up-regulation or down-regulation of mRNA levels as quantified by RT-qPCR are shown compared to that of the untreated cells, and those were normalized to the housekeeping gene B2M.

A graphical display of unique expression is observed in scatter plots where genes whose changes in expression exceeds thresholds of 2-fold induction or 0.5-fold reduction are shown individually for 8 and 24 h (Fig. 2).

Inflammatory response

Some of the genes included in the array employed here are intermediaries of inflammation, an immune response of vascular tissue to harmful stimuli, such as pathogens, irritants, or even damaged cells. The expression level of some of these genes changed after iron-cyclophane exposure. Among these is the ubiquitous nuclear transcription factor κB (NFκB), a pleiotropic regulator of many genes involved in immune and inflammatory processes (Conner & Grisham, 1996).

Most of the inflammatory changes in gene expression observed occur at the 24 h time point with either iron-cyclophane. Fe₂PC induced changes in the gene expression of members of the IL-1 family, CCR and TLR4 (Table 2). Importantly, pro-inflammatory IL1A was up-regulated early in the response at 8 h (2.20-fold) for Fe₂PO, and down-regulated later, after 24 h exposure. Fe₂PC exposure also evoked this expression pattern. Otherwise, pro-inflammatory IL1B was down-regulated early in the response at 8 h for both complexes, but after 24 h exposure for Fe₂PC, up-regulated expression was observed (2.25 fold).

Non-inflammatory immune response

This study assesses genes not only involved with inflammatory immune responses, but also genes related to other innate and adaptive immune responses. Modification of expression levels of several genes by Fe(III) complexes indicates that pattern recognition receptors can activate antigen-presentation and therefore stimulate T cell-mediated immune responses. These effects go beyond NFκB signaling and inflammation. Pattern recognition receptors belong to the innate response, such as the peptidoglycan recognition proteins (PGLYRP) family, which were induced by Fe₂PC (Table 2). CD14, along with Toll-like receptor protein (TOLLI) were down-regulated at 24 h for both metal complexes. TOLLIP interacts with TLR4 during recognition of the Gram-negative bacterial lipopolysaccharide (LPS). CD14 suppression could suggest an unexpected response of this receptor for the perception of moieties other than conserved LPS patterns in bacterial cell walls (Gilroy et al., 2004).

Apoptotic response

Genes related to the programed cell death pathway were interrogated to determine their responses to metal cyclophane treatment. The apoptotic gene for the adenosine receptor ADORA2A was strongly down-regulated by both Fe₂PO and Fe₂PC. Tumor necrosis factor TNF binds its receptor ligand to start the external apoptotic pathway, and down-regulation of the TNF receptor superfamily member 1A (TNFRSF1A) was also observed for both metal complexes after the 24 h treatment.

Nevertheless, NLRC4 was strongly up-regulated, suggesting the possibility of apoptosis after 24 h treatment. The caspase recruitment domain (CARD) containing protein 4 (NLRC4) interacts with caspase 1 and NOS2 (Damiano, Newman & Reed, 2004; Damiano, Oliveira & Welsh, 2004), and was strongly induced at 8 and 24 h by Fe₂PO and 24 h by Fe₂PC (Table 2). NFκB is essential for the transcriptional upregulation of these apoptotic pathways, and even though cytosolic levels of NFKB1 mRNA were upregulated after 24 h exposure, transcript levels of NFKB2 were down at both time points. It has been demonstrated in some conditions that NFκB acts as an anti-apoptotic protein, suggesting an unknown mechanism by which NFκB protects against cell death (Ghosh, May & Kopp, 1998).

Stress responses by reactive oxygen intermediates and/or metals

Since iron is complexed into the host molecules, we looked into stress response genes for changes in gene expression. The results show that neither iron-cyclophane complex led, to changes in expression of genes related to cellular stress such as HMOX1, IKBK and NFKBd. For all of these genes, down-regulated effects were observed for both Fe₂PO and Fe₂PC metal complexes, growing to a strong effect at the 24 h exposure time point.