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. Author manuscript; available in PMC: 2021 Aug 6.
Published in final edited form as: Immunobiology. 2016 Sep 5;222(2):399–408. doi: 10.1016/j.imbio.2016.08.010

Evaluation of a nanotechnology-based approach to induce gene-expression in human THP-1 macrophages under inflammatory conditions

Laura Bernal a,b,#, Abigail Alvarado-Vázquez a,#, David Wilson Ferreira a,c, Candler A Paige a,2, Cristina Ulecia-Morón a,d, Bailey Hill a, Marina Caesar a, E Alfonso Romero-Sandoval a,*
PMCID: PMC8344734  NIHMSID: NIHMS1728921  PMID: 27615510

Abstract

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5 μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes.

Keywords: Macrophages, THP-1 cells, Polyethylenimine, Inflammation, Gene-delivery, Cytokines

1. Introduction

Macrophages are key participants in many human physiological processes such as development, homeostasis, inflammation and immune responses. They are also critical in the pathophysiological mechanisms of conditions associated with chronic inflammation (i.e. diabetes, atherosclerosis, rheumatoid arthritis, chronic post-surgical pain, etc., (Wynn et al., 2013)). Macrophages are able to adapt to different conditions, and this plasticity allows them to adopt different phenotypes mostly to induce and then resolve inflammatory processes (Mills, 2012; Mosser, 2003).

Monocyte/macrophages actively participate in the early cytodestructive stage of inflammation and subsequently in its resolving phase by driving tissue regeneration (Leibovich and Ross, 1975). After tissue damage monocytes migrate to the site of injury, differentiate into macrophages, and acquire an M1 phenotype to initiate the inflammatory process. Then, macrophages drive the resolution of inflammation and promote tissue repair by switching their phenotype from M1 to M2 (Gordon and Martinez, 2010; Labonte et al., 2014; Mantovani et al., 2013). An abnormally prolonged M1 phenotype in macrophages may result in chronic inflammatory processes found in conditions such as chronic pain, atherosclerosis, obesity, etc. Recent studies have shown peripheral (muscle) M1 macrophages are associated in prolonged hyperalgesia in mice (Leung et al., 2015). Similarly, M1 macrophages in the spinal cord contribute to chronic pain in rodent model of spinal cord injury (Willemen et al., 2012; Kigerl et al., 2009). Moreover, the induction of an M2 macrophage phenotype reverses or prevents the development of pain and inflammation in rodent models of paw incision and diabetes (Saito et al., 2015), acid-induced muscle pain (Leung et al., 2015), carrageenan-induced inflammation (Willemen et al., 2012) or spinal cord injury (Willemen et al., 2012; Kigerl et al., 2009). In humans (Brueckmann et al., 2004) and mice (Khallou-Laschet et al., 2010), the extended presence of M1 macrophages and a systemic inflammatory response are associated with the development of atherosclerosis and acute coronary syndrome. The link between an M1 phenotype and insulin resistance has been demonstrated in both animal models and humans (Odegaard and Chawla, 2008). In a rodent model of obesity, the resolution of adipose inflammation by inducing M2 macrophages via resolvins reduced markers of chronic kidney disease and hepatic fat (Borgeson et al., 2015). Conversely, in some pathologies such as cancer, macrophages and other monocytic lineage cells abnormally acquire a persistent M2 or anti-inflammatory phenotype which allows tumor cells proliferation and metastasis (Qian and Pollard, 2010; Mantovani et al., 2002). Furthermore, this alternatively activated M2 cellular phenotype has been associated with poor survival in cancer patients (Petrillo et al., 2015; Lan et al., 2013; Curiel et al., 2004). Numerous studies have established the role of M2 macrophages in the pathophysiology of allergic airway diseases such as asthma, yet their function remains controversial (Balhara and Gounni, 2012). Thus, an abnormally persistent macrophage phenotype, M1 or M2, may determine either the resolution or the progression of certain pathological conditions. Thereby, the programmed and controlled induction of a specific macrophage phenotype is a viable therapeutic approach.

Cell-directed gene therapy using nanotechnology is a novel therapeutic strategy greatly focused on targeting immune cells (i.e. dendritic cells, macrophages, etc.). For example, dendritic cells have been successfully targeted by nanocomplexes in a murine model of ovarian cancer (Cubillos-Ruiz et al., 2009a, 2012). In these studies, polyethylenimine (PEI) nanoparticles complexed with specific small interference RNA (siRNA) or micro RNA (miRNA) effectively activated anti-tumoral responses, which blocked tumor progression and increased mice survival. Polyethylenimine nanoparticle is an endotoxin-free cationic polymer that protects DNA from degradation (Grzelinski et al., 2006; Urban-Klein et al., 2005). In fact, PEI nanoparticles have been recently used in clinical trials as gene delivery-vectors due to their proven stability and safety. In HIV patients, whose immune systems are unresponsive or deficient, PEI nanoparticles grafted with a mannose receptor were efficiently used to induce HIV antigens specifically in dendritic cells, which resulted in the induction of a robust immune response and a reduction of viral load for several months (Rodriguez et al., 2013; Lisziewicz et al., 2012). These approaches have taken advantage of the immunogenic responses of PEI (Cubillos-Ruiz et al., 2009b). This is logical, since under these conditions (cancer or AIDS) the desired effect is the induction of a more efficient immune response via specific gene induction. Whether these putative nanoparticle-induced immunogenic responses remain or alter gene induction under inflammatory conditions is still unclear. This study is designed to resolve whether this nanoparticle technology is suitable for a cell-directed gene therapy under conditions in which a reduction of an exacerbated immune response is needed (i.e. arthritis, peripheral neuropathies, atherosclerosis, obesity, etc.).

We conducted a series of experiments to test the hypothesis that PEI-derived nanoparticles effectively and safely induce gene overexpression without displaying an immunogenic pattern in macrophages with a pro-inflammatory phenotype. We based our hypothesis on the fact that macrophages under an M1 phenotype display a robust pro-inflammatory response that could render PEI immunogenicity insignificant or irrelevant. The use of lipopolysaccharide (LPS) allows us to induce an M1 phenotype in macrophages via toll-like receptor (TLR)-4, avoiding a competition with PEI nanoparticles that interact with TLR-5 (Cubillos-Ruiz et al., 2009b). A concomitant interaction of LPS and PEI nanoparticles on TLR-5 could mask potential immunogenic responses. Thus, we compared the potential toxicity, nanoparticle uptake, gene induction, and production of inflammatory factors by M1 macrophages in the presence or the absence of PEI-derived nanoparticles.

2. Materials and methods

2.1. Cell culture

Immortalized human acute monocytic leukemia cell line (THP-1) were plated in 75 cm2 flasks at 8 × 104 cells/cm2 with Roswell Park Memorial Institute 1640 media (RPMI 1640. Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were incubated at 37 °C in a 5% CO2 atmosphere throughout the experiment. THP-1 cells were a generous gift of Dr. Alan Eastman’s laboratory, Geisel School of Medicine at Dartmouth.

2.2. THP-1 cell differentiation and lipopolysaccharide (LPS) stimulation

THP-1 monocytes were plated at 1 × 106 cells/mL and were seeded in 75 cm2 flasks with RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. THP-1 monocytes were differentiated into THP-1 macrophages with 60 ng/mL phorbol-12-myristate-13-acetate (PMA, Sigma, St. Louis, MO) for 36 h (incubated at 37 °C under 5 % CO2) (Landry et al., 2012). Media was removed from the flasks, and 6 mL of Trypsin/EDTA (Mediatech Inc, Manassas, VA) was added for 5 min at 37 °C. The reaction was stopped using supplemented media and cells were centrifuged at 1200 rpm for 5 min, and re-suspended to a concentration of 250,000 cells/mL.

Cells were seeded on 24-well plates (250,000 cells/well) for 1 h and then stimulated with 5 μg/mL LPS (Escherichia coli O111:B4, Sigma) and incubated for 24–96 h depending on the experiment. When a second stimulus was necessary, supernatants were removed at 48 h. Then, fresh new media followed by a second challenge of LPS (5 μg/mL) was added, and cells were incubated in the same conditions for 4 and 24 h.

2.3. Nanoparticle preparation and cell transfection

For transfection, we used a commercially available nanoparticle (polyethylenimine, PEI) grafted with a mannose receptor ligand (Man-PEI; Polyplus Transfection, New York, NY). We complexed Man-PEI with a cDNA plasmid (using a pCMV6-XL4 vector) following the manufacturer’s instructions. Higher PEI nitrogen per DNA phosphate (N/P) ratios enhance the positive net charge of PEI-DNA complexes, which improves cell interaction and subsequently enhances the cellular and nuclear uptake and retention (Oh et al., 2002). The optimal PEI N/P ratio in murine fibroblasts is 10 (Boussif et al., 1995), and in monkey kidney cells is 5 (Thomas et al., 2005). We kept in mind that higher N/P ratios entail higher toxicity potentials (see Discussion for further details). To minimize cell toxicity and maximize gene induction efficiency in our preparation we use an N/P ratio of 5 as demonstrated elsewhere (Lisziewicz et al., 2001). Briefly, 1 μg of Man-PEI nanoparticle was mixed in 50 μL of NaCl Man-PEI (150 mM). This solution was mixed with either a GFP plasmid, CD14 plasmid, CD68 plasmid or an empty vector plasmid as negative control (pGFP, pCD14, pCD68 or pEmpty, respectively; 0.5 μg of plasmid in 50 μL of NaCl) and incubated for ~30 min at room temperature. Then, the 100 μL of Man-PEI + plasmid were added to cells simultaneously with LPS. The CD14, CD68, and empty vector plasmids were obtained from Origene (Rockville, MD). The GFP plasmid was obtained from Biorad (Hercules, CA). Supernatants or cells were collected at different time points for further studies (see details below).

2.4. Nanoparticle cell uptake and gene induction efficacy

To measure the efficacy of nanoparticle cellular uptake, Man-PEI was complexed, as previously described, with an siRNA labeled with a red fluorophore (Alexa 555, 2 μg) or an unlabeled negative siRNA control (2 μg). To measure gene induction efficiency, Man-PEI was complexed with a plasmid encoding GFP (pGFP) or empty vector (pEmpty), which allowed the quantification of positively transfected cells. First, a rounded coverslip (12 mm diameter) was placed into the wells of a 12-well plate. Fibronectin (500 μg/μL, Sigma) was added to each well and incubated for 12 h at 4 °C. After fibronectin was washed with sterile PBS, cells were seeded on 12-well plates (250,000 cells/well) containing the prepared coverslips for 1 h and then treated with LPS and Man-PEI complexes as described above for 48 h. Following incubation, supernatants were removed and the coverslips containing the attached cells were incubated with DAPI (1:1000 in PBS, 5 min) to mark cellular nuclei.

Fluorescence microcopy was used to quantify Alexa 555-labeled Man-PEI cellular uptake and GFP protein expression in the cells 48 h after cell transfection. Images shown were acquired using a Leica DMIL microscope (Model: 11521258) and a Leica DFC345 FX Digital Camera (Leica Microsystems Inc., Buffalo Grove, IL). The proportion of positive fluorescent cells was calculated in both Alexa 555 and control (non-labeled) siRNA groups, using DAPI stained cells as total cell population in our preparations. For these particular experiments the red fluorescence of Alexa 555 was changed to green for better visual representation. Quantitative analysis of the immunofluorescence intensity of GFP was performed using Sigma Scan Pro software (Systat Software Inc., San Jose, CA).

2.5. Cytotoxicity

We performed lactate dehydrogenase (LDH) and trypan blue assays to measure cell viability in THP-1 macrophages upon different conditions: cells without stimulation (control), cells treated with Man-PEI, cells stimulated with LPS, and cells stimulated with LPS in the presence of Man-PEI.

Cells of the abovementioned groups were plated in a 96-well plate for LDH assay. After 24 and 48 h of incubation, the plate was centrifuged at 400 rpm, 4 °C for 10 min, and then 100 μL of supernatant was transferred to a new plate to measure the levels of LDH leakage. We used a commercially available LDH assay kit (Clontech Lab Inc., Mountain View, CA) following the manufacturer’s instructions. The plate was read in a Culture DTX880 Multimode Detector (Beckman Coulter Inc., Fullerton, CA) using a 450 nm excitation filter. As positive controls for LDH leakage we used different cell concentrations treated with 2% Triton X (Alfa Aesar, Ward Hill, MA). The cell concentrations for these positive controls were 1250/mL, 2500/mL, 5,000/mL, 10000/mL, 20000/mL, and 40000/mL. Using 2% Triton X in different cell concentrations we confirmed that the sensitivity of the LDH leakage is set at 1250 cells/mL (data not shown). This approach allowed us to compare the degree of LDH production (cell death) in all groups.

For trypan blue assay, the same groups were plated in a 24-well plate and incubated for 48 h, the time in which we observed increased LDH production. This approach allowed us to determine the percent of viable cells in our different conditions. Forty-eight hours after stimulation and treatment, the supernatant was then removed and fresh media with trypan blue dye (600 μL, proportion 1:1, Life Technologies, Grand Island, NY) was added to each well for one minute at 37 °C. Cells were counted using a hemocytometer (VWR International, Philadelphia, NJ). Dead and viable cells were counted under a light microscope by counting three random fields using a 20× objective, with dead cells identified as trypan blue positive cells. The percentage of cell viability was then calculated.

2.6. RNA isolation and quantitative real time (RT)-PCR

Following cell incubation (from 24 to 96 h after single stimulation, or 4 and 24 h after double stimulation), cells were washed with 1 mL of ice-cold sterile PBS, collected using BL + TG buffer (PBS and 1-thioglycerol), and stored at −80 °C until RNA isolation experiments. RNA was isolated from THP-1 macrophages using ReliaprepTM RNA Cell Miniprep System (Promega, Madison, WI) according to manufacturer’s protocols.

Levels of mRNA were determined as described previously (Ndong et al., 2012). Briefly, 1 μg of total RNA from each sample was reverse transcribed into cDNA using the ScriptTM Reverse Transcription Supermix (BioRad, Hercules, CA) in the following conditions: 5 min at 25 °C, 30 min at 42 °C and 5 min at 85 °C. We quantified the expression of TNF-α (59 °C), IL-6 (60 °C), IL-10 (59 °C), CD14 (57 °C), CD68 (58 °C), GFP (60 °C) and β-actin (57 °C) using the SsoAdvancedTM Universal SYBR Green Supermix (BioRad, Hercules, CA) in the following conditions: 1 cycle of 98 °C for 30 s, 45 cycles of 98 °C for 15 s followed by 30 s of the primer-specific annealing temperature. The primers for RT-PCR are shown in Table 1. All samples were run in duplicate using the CFX96 Real-Time PCR system (BioRad, Hercules, CA). A melt curve analysis was performed between 65 °C and 95 °C in 0.5° intervals (5 s per interval). The expression of mRNA for our molecules of interest was normalized to β-actin expression level. The fold change of each gene was determined using the ddCt method as previously described (Livak and Schmittgen, 2001). All data were further normalized to the respective control (non-stimulated cells at 0 h or pEmpty group at 24 h, when appropriate) that was given a value equal to 1.

Table 1.

Primers for RT-PCR analyses.

PCR Primers Forward (5’−3’) Reverse (5’−3’)
IL-6 ATG CAA TAA CCA CCC CTG AC GAG GTG CCC ATG CTA CAT TT
IL-10 CAT CGA TTT CTT CCC TGT GAA TCT TGG AGC TTA TTA AAG GCA TTC
TNF-α CCC AGG GAC CTC TCT CTA ATC ATG GGC TAC AGG CTT GTC ACT
CD14 GCT GGA CGA TGA AGA TTT CC ATT GTC AGA CAG GTC TAG GC
CD68 GCT ACA TGG CGG TGG AG ACA A ATG ATG AGA GGC AGC AAG ATG G
GFP CGA GTT TGT GTC CGA GAA TG GGT GTT CAA TGC TTT TCC CG
β-actin AGA GCT ACG AGC TGC CTG AC AGC ACT GTG TTG GCG TAC AG
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2.7. ELISA assays

The concentration of TNF-α, IL-6 and IL-10 in supernatants were measured with commercial sandwich enzyme-linked immunosor-bent Assay kits (Human TNF-α/IL-10/IL-6 ELISA Ready-SET-Go!, eBioscience, San Diego, CA). Sensitivity of the ELISA kits is as follows: 4 pg/mL for human TNF-α and 2 pg/mL for human IL-6 and IL-10 kits. ELISA assays were performed following the manufacturer’s instructions to measure the cytokine release at 0 h (control), and from 24 to 96 h after single stimulation, or 4 and 24 h after double stimulation. All the data were normalized to the control group (0 h), which was given a value equal to 1. When protein concentration in the control group was below the ELISA kit sensitivity (non-detected), the lowest standard value (sensitivity level) of the respective ELISA assay was used for normalization and statistical analysis.

2.8. Statistical analysis

All experiments were completed at least two times, in duplicates. Statistical analysis was performed using GraphPad Prism 6.01 (GraphPad Software Inc., CA). Unpaired t-test, one- or two-way ANOVA followed by Bonferroni’s post hoc test was used as appropriate. A P < 0.05 was considered significant. Data are presented as mean ± standard deviation (SD).

3. Results

3.1. Man-PEI cell uptake efficacy and gene induction efficiency

The incubation (48 h) of THP-1 macrophages with Man-PEI plus an Alexa 555-labeled siRNA resulted in a significant cellular uptake efficacy (69.42 ± 6.22% fluorescence positive cells, Fig. 1) in comparison with cells incubated with the control siRNA (18.25 ± 2.57%, potential autofluorescence, image not shown). As shown in Fig. 1, we observed abundant cytoplasmic and peri-nuclear presence of nanoparticles, as well as intra-nuclear transfection (Fig. 1, right lateral image). Red fluorescence of Alexa 555 was changed to green in Fig. 1 for better visual representation.

Fig. 1.

Fig. 1.

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Cellular transfection of Man-PEI in THP-1 macrophages. Microscopic images of THP-1 macrophages with intracellular Man-PEI complexed with a siRNA labeled with Alexa 555 using Man-PEI. Alexa 555’s red fluorescence was changed to green for better visualization of the nanoparticle cellular uptake. The inserted magnified image shows robust cytoplasmic and nuclear presence of fluorescence, indicative of Man-PEI transfection. The magnified right image displays a detail of nuclear fluorescence, indicative of Man-PEI transfection.

To test the gene induction efficiency of Man-PEI, THP-1 macrophages were incubated (24 and 48 h) with a GFP plasmid (pGFP) or an empty vector (pEmpty) complexed with Man-PEI. We observed a significant increase of GFP mRNA at 24 and 48 h (Fig. 2). We also detected GFP positive cells by immunocytochemistry and fluorescence microscopy at the time of maximum mRNA expression (48 h). The efficiency of Man-PEI-induced GFP protein expression reached 82 ± 4.72% of cells in the pGFP group, compared with 28 ± 2.43% cells with green florescence (probably autofluorescence) in the pEmpty vector group (Figs. 3 (a) and Fig. 3(b)). The average fluorescence intensity per pixel was higher in the pGFP group than in the pEmpty group (Fig. 3(c)).

Fig. 2.

Fig. 2.

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Efficiency of GFP gene induction with Man-PEI in THP-1 macrophages. Quantification for GFP mRNA induction using Man-PEI complexed with pGFP in LPS-stimulated THP-1 macrophages in comparison to cells with LPS plus Man-PEI complexed with pEmpty (pEmpty), cells with LPS plus unconjugated Man-PEI, cells with LPS alone, or cells with no treatment incubated for 24 and 48 h. N = 4–6 per group. The expression of GFP mRNA was normalized to the respective levels of β-actin expression in each group, and then calculated as fold change against the expression of pEmpty group at 24 h. *p < 0.05 vs. pEmpty; # p < 0.05, 24 h vs. 48 h by Two way ANOVA followed by Bonferroni post-hoc test.

Fig. 3.

Fig. 3.

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GFP protein induction using Man-PEI. (a) Microscopic images of nuclear staining using DAPI (blue in left image) and GFP protein fluorescence (green, right image) in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmpty) or a plasmid encoding for GFP (pGFP) at 48 h. Co-regionalization (acqua/bluish-green) of DAPI and GFP is observed in the right image. (b) Quantification (percent of total cells stained with DAPI, blue) for green fluorescent cells in LPS-stimulated THP-1 macrophages transfected with Man-PEI + pEmpty or Man-PEI + pGFP at 48 h (N = 3 per group). (c) Quantification for the average intensity of green fluorescence (arbitrary units) in LPS-stimulated THP-1 macrophages transfected with Man-PEI + pEmpty or Man-PEI + pGFP at 48 h (N = 3 per group). *P < 0.05 vs. pEmpty by student’s t-test.)

3.2. Lack of cytotoxicity of Man-PEI

No significant differences were observed in LDH release levels in Man-PEI, LPS or LPS + Man-PEI groups when compared to the non-stimulated control group at 24 and 48 h after incubation (Fig. 4(a)). Forty-eight hours after incubation we found an increase of LDH release among all groups in comparison with their release at 24 h, which may reflect the natural cell death rate under normal conditions.

Fig. 4.

Fig. 4.

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Cell viability of THP-1 macrophages using Man-PEI. (a) LDH release quantification in THP-1 macrophages under control conditions (no treatment and no LPS; Control) in comparison to cells with unconjugated Man-PEI alone (no LPS; Man-PEI), cells with LPS alone (no other treatment; LPS) or cells with LPS plus unconjugated Man-PEI (LPS + Man-PEI) incubated for 24 and 48 h. All values were normalized to the control group at 24 h (N = 9 per group). *p < 0.05, vs. 24 h by Two-way ANOVA followed by Bonferroni’s post-hoc test. (b) Quantification for THP-1 macrophages positively stained with Trypan Blue in control, Man-PEI, LPS alone, and LPS + Man-PEI groups incubated for 48 h. All values were normalized to the control group (N = 6 per group).

Consistent with the LDH assay, trypan blue exclusion assays showed that cell death among the different groups (Man-PEI alone, LPS or LPS + Man-PEI) was low and similar to the control group (Fig. 4(b)). In addition, we did not find changes in the percentage of cell death 96 h after a single LPS stimulation among the control (10.6 ± 2.2%), Man-PEI (10.8 ± 3.8%), LPS (9.9 ± 3.1%) and LPS + Man-PEI (11.2 ± 3.5%) groups. Similarly, 24 h after a second LPS stimulation the percentage of death cells in the Man-PEI (6.5 ± 3.0%), LPS (7.5 ± 2.5%) or LPS + Man-PEI (6.7 ± 2.3%) group was similar to the control group (6.0 ± 3.8%). This confirms that LDH increase at 48 h vs. 24 h is due to the normal cell death rate under culture conditions, and that Man-PEI does not induce cytotoxicity in non-stimulated and LPS-stimulated cells under our experimental conditions.

3.3. Effects of Man-PEI on cytokine release

The effects of the nanoparticle on cytokines profile at mRNA and protein levels were determined following a single LPS stimulation at incubation times of 24, 48, 72 and 96 h. The stimulation with LPS produced an increase in TNF- α, IL-6 and IL-10 protein at all the studied time points (Table 2). As shown in Fig. 5, TNF-α mRNA was reduced in LPS + Man-PEI (normalized to 0 h) when compared to the LPS alone group at 72 h (Fig. 5(a)), whereas TNF-α protein concentration (Fig. 5(d)) was not significantly different in LPS + Man-PEI and LPS alone groups. We found a down-regulation of IL-6 mRNA in the LPS + Man-PEI group when compared to the LPS alone group at 48, 72 and 96 h (Fig. 5(b)). At the protein level, IL-6 was significantly lower at 24 h in the LPS + Man-PEI group when compared to the LPS alone group, but no differences were observed between groups at 48, 72 and 96 h (Fig. 5(e)). We observed that the mRNA levels of IL-10 did not differ from the levels found in LPS alone group (Fig. 5(c)). At the protein level, we observed that IL-10 concentration slightly decreased in LPS + Man-PEI group when compared to the LPS alone group at 72 h, but not at 24, 48 and 96 h (Fig. 5(f)). In summary, mild reductions in the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 at the protein level were observed in the LPS + Man-PEI group when compared to the LPS alone group.

Table 2.

Time course of cytokine release following a single stimulus with LPS.

0 h 24 h 48 h 72 h 96 h
TNF-α 88.1 ± 34.4 167.5 ± 20.2* 467.1 ± 247.5* 238.2 ± 20.5* 269.1 ± 6.3*
IL-6 N.D. 16.6 ± 1.6* 141.3 ± 52.2* 28.9 ± 1.7* 41.4 ± 0.7*
IL-10 N.D. 26.3 ± 0.9* 33.0 ± 4.15* 143.3 ± 5.3* 88.7 ± 16.8*
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Times are presented in h (h). Values of cytokines are shown in pg/mL. N.D. = non-detected.

*

P < 0.05, vs. 0 hour by Student’s t-test.

Fig. 5.

Fig. 5.

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Changes of cytokine expression (mRNA and protein release) in Man-PEI transfected macrophages following a single LPS stimulus. Quantification for TNF-α (a), IL-6 (b) and IL-10 (c) mRNA, and TNF-α (d), IL-6 (e) and IL-10 (f) protein concentration in THP-1 macrophages stimulated with LPS in the presence or absence of Man-PEI (LPS + Man-PEI and LPS respectively) at 24, 48, 72 and 96 h. The expression of mRNA was normalized to the respective β-actin expression and then, values were further normalized to the control group (0 h) which was assigned a value equal to 1. (N = 3–7 per group). The protein concentration was normalized to the control group (0 h) which was assigned a value equal to 1. (N = 3–9 per group). The mRNA expression and protein concentration of all cytokines in the LPS group were significantly different from the control group at all the studied time points (p < 0.05, vs. control by student’s t-test; significance symbols not shown for clarity). *p < 0.05, vs. LPS by student’s t-test.

We also investigated the effect of Man-PEI in THP-1 macrophages stimulated with a double challenge of LPS (applied 48 h after the first stimulus). An increase in the concentration of TNF-α, IL-6 and IL-10 were observed at 4 and 24 h after the second LPS stimulus (+4 and +24, respectively, Table 3). We compared the mRNA and protein concentration of the abovementioned cytokines at +4 and +24 h in the presence and absence of Man-PEI nanoparticle. We did not find changes in TNF-α mRNA (Fig. 6(a)) or protein concentration (Fig. 6(d)) between LPS + Man-PEI and LPS alone groups at any studied time point. We did not observe changes in IL-6 mRNA between LPS + Man-PEI and LPS alone groups at +4 or +24 h (Fig. 6(b)). However, we found a reduction in IL-6 at the protein level in the LPS + Man-PEI group when compared to the LPS alone group at +4 h (Fig. 6(e)), and no changes were observed at +24 h. For IL-10, we found a reduction at the mRNA (Fig. 6(c)) and protein (Fig. 6(f)) level in the LPS + Man-PEI group when compared to the LPS alone group at +4 h, and no change was observed at +24 h. In summary, reductions in both the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 were observed at the protein level in the Man-PEI + LPS group when compared to the LPS alone group only at +4 h.

Table 3.

Time course of cytokine release following double stimulation with LPS.

0 h +4 h +24 h
TNF-α 88.1 ± 34.4 173.8 ± 89.6* 10.57 ± 2.4*
IL-6 N.D. 17.35 ± 5.7* 74.1 ± 14.5*
IL-10 N.D. 5.8 ± 3.9* 196.0 ± 96.5*
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Times are presented in hours (h). Values of cytokines are shown in pg/mL. N.D. = non-detected.

*

P < 0.05, vs. 0 hour by Student’s t-test.

Fig. 6.

Fig. 6.

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Changes of cytokine expression (mRNA and protein release) in Man-PEI transfected macrophages challenged with a second stimulus of LPS. Quantification for TNF-α (a), IL-6 (b) and IL-10 (c) mRNA, and TNF-α (d), IL-6 (e) and IL-10 (f) protein concentration in THP-1 macrophages with a double stimulation with LPS in the presence or absence of Man-PEI (LPS + Man-PEI and LPS respectively) at 4 and 24 h after the second stimulus (+4 and +24 respectively). The expression of mRNA was normalized to the respective β-actin expression and then, values were further normalized to the control group (0 h) which was assigned a value equal to 1. (N = 4–6 per group). The protein concentration was normalized to the control group (0 h) which was assigned a value equal to 1. (N = 5–13 per group). The mRNA expression and protein concentration of all cytokines in the LPS group were significantly different from the control group at all the studied time points (p < 0.05, vs. control by student’s t-test; significance symbols not shown for clarity). *p < 0.05, vs. LPS by student’s t-test.

3.4. Man-PEI for induction of constitutively expressed genes

To further confirm the gene induction efficiency of Man-PEI we tested this approach using plasmids of genes that are normally and constitutively expressed in human macrophages: pCD14 and pCD68. We observed an increase in CD14 mRNA at 24 h after Man-PEI transfection (Fig. 7(a), inserted graph). At 48 h the expression of CD14 mRNA increased in all groups. However, the over-expression of CD14 mRNA in pCD14-trasfected macrophages was significantly higher (~4 × 107-fold increase) when compared to pEmpty-transfected cells (Fig. 7(a)). Similarly, we observed an over-expression of CD68 mRNA at 24 and 48 h in pCD68-transfected macrophages, with a peak in gene induction at 48 h (over 700-fold increase) when compared to pEmpty-transfected cells (Fig. 7(b)).

Fig. 7.

Fig. 7.

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CD14 and CD68 mRNA induction using Man-PEI. Quantification for CD14 (a) and CD68 (b) mRNA induction using Man-PEI complexed with either pCD14 or pCD68 in LPS-stimulated THP-1 macrophages in comparison to cells with LPS plus Man-PEI complexed with pEmpty (pEmpty), cells with LPS plus unconjugated Man-PEI, cells with LPS alone, or cells with no treatment incubated for 24 and 48 h. The inserted magnified graph in (a) shows the different expression of CD14 following 24 or 48 h of transfection. N = 3–7 per group. The expression of CD14 and CD68 mRNA was normalized to the respective levels of β-actin expression in each group, and then calculated as fold change against the expression of pEmpty group at 24 h. *p < 0.05 vs. pEmpty; # p < 0.05, 24 h vs. 48 h by Two way ANOVA followed by Bonferroni post-hoc test.

4. Discussion

The main findings of our study are: (1) Man-PEI nanoparticles showed high efficacy and efficiency to induce gene expression in THP-1 macrophages under inflammatory conditions without overt cytotoxic effects; and (2) Man-PEI themselves slightly and transiently modify the production of both pro- and anti-inflammatory cytokines in M1 THP-1 macrophages without inducing a clear immunogenic response.

The ability of PEI or PEI-derivatives to promote gene induction in vitro and in vivo is well documented (Urban-Klein et al., 2005; Cubillos-Ruiz et al., 2009b; Godbey et al., 1999a). Boussif et al. (1995) demonstrated the efficiency of generic PEI for in vitro gene induction in a variety of cell lines (mouse fibroblasts, monkey kidney and human hepatoma, human leukemia, human epithelial carcinoma and human lung epithelium, and primary cell cultures from rat endothelial cells). Moreover, they demonstrated that luciferase expression using in vivo PEI transfection in chicken embryonic neurons was similar to that obtained in primary brain cells in vitro at the optimal N/P ratio of 9. These data are also in accordance with previous data from our laboratory in which in vivo gene induction using PEI was demonstrated in rat spinal cord in a model of neuropathic pain (Ndong et al., 2012).

Cell-binding ligands have been used to attempt to improve the efficiency of the PEI nanoparticle transfection, and this enables ligand-receptor mediated endocytosis. Herein, we used Man-PEI, a modified PEI nanoparticle grafted with 3% mannose. This approach not only improves the efficiency of gene delivery when compared to generic PEI, but also provides cell selectivity since this mannosylated nanoparticle preferentially targets cells expressing mannose receptors (Jiang et al., 2009). Mannose receptors are expressed in cells of monocytic lineage, such as macrophages and dendritic cells, and they are considered a hallmark of immature monocyte-derived cells (Martinez-Pomares, 2012). Thus, Man-PEI specifically targets macrophages, as it has been shown elsewhere using murine Raw 264.7 cells vs. HeLa cells (Jiang et al., 2009). It has been described that generic PEI of high molecular weight (800 kDa) using an N/P ratio of 9 produces 100% cellular/nuclear transfection rates in chicken neurons (Boussif et al., 1995). Herein, we observed that Man-PEI (22 kDa nanoparticle, N/P = 5) was found in the cell cytosol and in the cellular nucleus in approximately 69% of cells, confirming the efficacy of our experimental conditions in M1 human macrophages. This lower (yet sufficient for gene induction) transfection rate could be explained by the differential N/P ratio and lower molecular weight of our nanoparticle when compared to the one used in the abovementioned study (Boussif et al., 1995). This level of cell and nucleus penetration strongly supports the efficiency of gene delivery and gene over-expression observed in our condition.

One potential concern of a high Man-PEI efficacy is the potential toxicity. Our data demonstrate that our Man-PEI nanoparticle under our experimental conditions did not produce cytotoxicity. This could be explained by the intrinsic characteristics of this particular nanoparticle and the specific conditions in which we prepared them for our studies. The efficacy of PEI-vectors depends upon different parameters, like a linear or branch structure, the molecular weight, the DNA content and the particle size. For example, high weight nanoparticles lead to higher transfection rates than low-weight nanoparticles, but their cytotoxicity is also higher (Kunath et al., 2002; Fischer et al., 1999; Godbey et al., 1999b). The molecular weight of our nanoparticle is 22 kDa-PEI, a weight that has not been associated with cytotoxic effects (Bonnet et al., 2008; Mizrahi et al., 2009). This is contrast to studies of biocompatibility between human fibroblast and osteoclasts and higher molecular weights PEI (i.e. 70 kDa) which produced a notable cytotoxic effect (Brunot et al., 2007). Importantly, our studies demonstrate that an N/P ratio of 5 provides excellent cellular and nuclear penetration as well as gene induction without no toxicity. This concurs with previous observations using other types of cells (Thomas et al., 2005). It is worth mentioning that these findings were obtained under inflammatory conditions, which could make these cells more vulnerable due to the exposure of multiple cytotoxic factors. Therefore, our data provides novel evidence in favor of a wider spectrum for Man-PEI use. We confirmed that Man-PEI efficiently induces an overexpression of GFP with a peak of gene induction at 48 h. Similarly, other genes that are regularly expressed by macrophages, namely CD14 and CD68, were efficiently overexpressed using Man-PEI under the same inflammatory conditions using LPS. The use of multiple controls and multiple analyses demonstrates the specificity of this approach (Fig. 7).

Lipopolysaccharide is widely used as a potent inductor of cytokine release in monocytes-derived cells by activating TLR-4 (Qureshi et al., 1999; Poltorak et al., 1998). THP-1 cells stimulated with LPS produce an orchestrated inflammatory response that begins with the release of pro-inflammatory cytokines (i.e. TNF-α, IL-1, IL-6) and is followed by the release of anti-inflammatory factors (i.e. IL-10, IL-4, etc.) that resolve the inflammatory process (Landry et al., 2012; de Waal Malefyt et al., 1991; Chanput et al., 2010) (Table 2). Using a single LPS stimulus we observed that Man-PEI produced a down-regulation of TNF-α and IL-6 mRNA as well as a slight reduction of both the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10 at the protein level. In an attempt to promote a sub-acute inflammatory condition, we applied two separate LPS stimuli at 0 and 48 h of incubation and tested the potential immunogenic responses of Man-PEI. Under these sub-acute inflammatory conditions, Man-PEI also induced a mixed immunogenic response at the protein level: reduction of pro- (IL-6) and anti-inflammatory (IL-10) cytokines. These data are in accordance with previous studies that showed that systemic administration of PEI complexed with DNA or siRNA in mice did not show major production of serum pro-inflammatory cytokines (i.e. no significant changes in TNF-α, IL-12, IL-6, slight change in interferon gamma), neither produced hepatic toxicity (Bonnet et al., 2008). Similarly, our data suggest that Man-PEI could produce a slight and transient immunogenic reaction in human macrophages under sub-acute inflammatory conditions, which could be explained by direct actions on TLR-5 (Cubillos-Ruiz et al., 2009b). It is likely that the presence of a mannose ligand in our nanoparticle plays a favorable role in minimizing PEI immunogenic effects since the activation of mannose receptors affects the production of cytokines upon an anti-inflammatory profile (i.e. increased production of IL-10) in LPS-maturing dendritic cells (Chieppa et al., 2003). This is of special relevance for the use of Man-PEI to induce anti-inflammatory genes in sub-acute inflammatory conditions, since it is possible that the activation of mannose receptors under these conditions favors the restoration of a more homeostatic state. Notwithstanding, our findings indicate that Man-PEI-induced immunogenic response is rather minimal and transient under inflammatory conditions in human macrophages.

The suitability of generic PEI complexed with nucleic acids has been previously confirmed in a therapeutic context in which an enhancement of the immune response was required. The induction of the micro RNA, pre-miR-155 in dendritic cells in a murine model of ovarian cancer promoted a more efficient immune response that resulted in a tumor size reduction (Cubillos-Ruiz et al., 2012). A similar effect was observed in this murine model using PEI and small interfering RNA (siRNA) (Cubillos-Ruiz et al., 2009b). Regarding Man-PEI, phase I/II clinical trials have been performed to induce HIV genes expression in monocytes to enhance the immune response against the virus. Specifically, a plasmid encoding for 15 HIV proteins (including a non-functional mutant integrase and truncated non-functional Nef) was complexed with Man-PEI and administered to HIV+ patients transdermally. This Man-PEI vaccine-like approach specifically targeted dendritic cells that subsequently activated naïve CD4+ and CD8+ T cells and reduced the viral load in patients for 48–61 weeks (Rodriguez et al., 2013; Lisziewicz et al., 2012). Even though these effects were long lasting, the peak response with three vaccinations was observed after 17 weeks of treatment, and the presence of mild side effects was transitory (Rodriguez et al., 2013). These clinical trials were preceded by in vitro experiments that demonstrated the feasibility of this cell-directed gene therapy to induce HIV genes using Man-PEI and dendritic cells as specific targets [33]. We now demonstrate that this technology could be useful under conditions of an over-responsive immune state. We postulate that this cell-directed gene therapy is suitable to induce specific genes that restore a homeostatic state under inflammatory conditions.

5. Conclusions

Our current data demonstrate that Man-PEI can efficiently and safely induce gene overexpression in human macrophages with M1 pro-inflammatory phenotype. Since macrophages have an active role in the induction and resolution of inflammation, they are attractive targets for cell-directed gene immunotherapies which aim to restore a homeostatic state under chronic inflammatory conditions. The clinical significance of our studies lies in the fact that targeting human monocytes using Man-PEI nanoparticles has proven to be effective, efficient and safe in patients. Furthermore, a transdermal delivery system has been used in Man-PEI clinical trials, which enhances the translational potential of our current findings.

Acknowledgments

The authors would like to acknowledge Rita Allen Foundation and American Pain Society- Pain Scholar Award (EAR-S), NIH-NIGMS R15GM109333 (EAR-S), Pharmacy Research Summer Internship (LB, AA, DWF, BH, ML) for funding, and CAPES Foundation − Ministry of Education of Brazil, PDSE: BEX 10794/14-0 for funding DWF’s scholarship.

Footnotes

Conflict of interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

REFERENCE LIST

  1. Wynn TA, Chawla A, Pollard JW, 2013. Macrophage biology in development, homeostasis and disease. Nature 496 (7446), 445–455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Mills CD, 2012. M1 and M2 macrophages: oracles of health and disease. Crit. Rev. Immunol 32 (6), 463–488. [DOI] [PubMed] [Google Scholar]
  3. Mosser DM, 2003. The many faces of macrophage activation. J. Leukoc. Biol 73 (2), 209–212. [DOI] [PubMed] [Google Scholar]
  4. Leibovich SJ, Ross R, 1975. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am. J. Pathol 78 (1), 71–100. [PMC free article] [PubMed] [Google Scholar]
  5. Gordon S, Martinez FO, 2010. Alternative activation of macrophages: mechanism and functions. Immunity 32 (5), 593–604. [DOI] [PubMed] [Google Scholar]
  6. Labonte AC, Tosello-Trampont AC, Hahn YS, 2014. The role of macrophage polarization in infectious and inflammatory diseases. Mol. Cells 37 (4), 275–285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mantovani A, Biswas SK, Galdiero MR, Sica A, Locati M, 2013. Macrophage plasticity and polarization in tissue repair and remodelling. J. Pathol 229 (2), 176–185. [DOI] [PubMed] [Google Scholar]
  8. Leung A, Gregory NS, Allen LH, Sluka KA, 2015. Regular physical activity prevents chronic pain by altering resident muscle macrophage phenotype and increasing IL-10 in mice. Pain. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Willemen HL, Huo XJ, Mao-Ying QL, Zijlstra J, Heijnen CJ, Kavelaars A, 2012. MicroRNA-124 as a novel treatment for persistent hyperalgesia. J. Neuroinflammation 9, 143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG, 2009. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J. Neurosci 29 (43), 13435–13444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Saito T, Hasegawa-Moriyama M, Kurimoto T, Yamada T, Inada E, Kanmura Y, 2015. Resolution of inflammation by resolvin D1 is essential for peroxisome proliferator-activated receptor-gamma-mediated analgesia during postincisional pain development in type 2 diabetes. Anesthesiology. [DOI] [PubMed] [Google Scholar]
  12. Brueckmann M, Bertsch T, Lang S, Sueselbeck T, Wolpert C, Kaden JJ, Jaramillo C, Huhle G, Borggrefe M, Haase KK, 2004. Time course of systemic markers of inflammation in patients presenting with acute coronary syndromes. Clin. Chem. Lab. Med 42 (10), 1132–1139. [DOI] [PubMed] [Google Scholar]
  13. Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston AT, Clement M, Dussiot M, Levillain O, Graff-Dubois S, Nicoletti A, Caligiuri G, 2010. Macrophage plasticity in experimental atherosclerosis. PLoS One 5 (1), e8852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Odegaard JI, Chawla A, 2008. Mechanisms of macrophage activation in obesity-induced insulin resistance. Nat. Clin. Pract. Endocrinol. Metab 4 (11), 619–626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Borgeson E, Johnson AM, Lee YS, Till A, Syed GH, Ali-Shah ST, Guiry PJ, Dalli J, Colas RA, Serhan CN, Sharma K, Godson C, 2015. Lipoxin A4 attenuates obesity-induced adipose inflammation and associated liver and kidney disease. Cell Metab. 22 (1), 125–137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Qian BZ, Pollard JW, 2010. Macrophage diversity enhances tumor progression and metastasis. Cell 141 (1), 39–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A, 2002. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23 (11), 549–555. [DOI] [PubMed] [Google Scholar]
  18. Petrillo M, Zannoni GF, Martinelli E, Pedone Anchora L, Ferrandina G, Tropeano G, Fagotti A, Scambia G, 2015. Polarisation of tumor-associated macrophages toward M2 phenotype correlates with poor response to chemoradiation and reduced survival in patients with locally advanced cervical cancer. PLoS One 10 (9), e0136654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lan C, Huang X, Lin S, Huang H, Cai Q, Wan T, Lu J, Liu J, 2013. Expression of M2-polarized macrophages is associated with poor prognosis for advanced epithelial ovarian cancer. Technol. Cancer Res. Treat 12 (3), 259–267. [DOI] [PubMed] [Google Scholar]
  20. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L, Zou W, 2004. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med 10 (9), 942–949. [DOI] [PubMed] [Google Scholar]
  21. Balhara J, Gounni AS, 2012. The alveolar macrophages in asthma: a double-edged sword. Mucosal Immunol. 5 (6), 605–609. [DOI] [PubMed] [Google Scholar]
  22. Cubillos-Ruiz JR, Fiering S, Conejo-Garcia JR, 2009a. Nanomolecular targeting of dendritic cells for ovarian cancer therapy. Future Oncol. 5 (8), 1189–1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Cubillos-Ruiz JR, Baird JR, Tesone AJ, Rutkowski MR, Scarlett UK, Camposeco-Jacobs AL, Anadon-Arnillas J, Harwood NM, Korc M, Fiering SN, Sempere LF, Conejo-Garcia JR, 2012. Reprogramming tumor-associated dendritic cells in vivo using miRNA mimetics triggers protective immunity against ovarian cancer. Cancer Res. 72 (7), 1683–1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Grzelinski M, Urban-Klein B, Martens T, Lamszus K, Bakowsky U, Hobel S, Czubayko F, Aigner A, 2006. RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts. Hum. Gene Ther 17 (7), 751–766. [DOI] [PubMed] [Google Scholar]
  25. Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A, 2005. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther. 12 (5), 461–466. [DOI] [PubMed] [Google Scholar]
  26. Rodriguez B, Asmuth DM, Matining RM, Spritzler J, Jacobson JM, Mailliard RB, Li XD, Martinez AI, Tenorio AR, Lori F, Lisziewicz J, Yesmin S, Rinaldo CR, Pollard RB, 2013. Safety, tolerability, and immunogenicity of repeated doses of dermavir, a candidate therapeutic HIV vaccine, in HIV-infected patients receiving combination antiretroviral therapy: results of the ACTG 5176 trial. J. Acquir. Immune Defic. Syndr 64 (4), 351–359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lisziewicz J, Bakare N, Calarota SA, Banhegyi D, Szlavik J, Ujhelyi E, Toke ER, Molnar L, Lisziewicz Z, Autran B, Lori F, 2012. Single dermaVir immunization: dose-dependent expansion of precursor/memory T cells against all HIV antigens in HIV-1 infected individuals. PLoS One 7 (5), e35416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Cubillos-Ruiz JR, Engle X, Scarlett UK, Martinez D, Barber A, Elgueta R, Wang L, Nesbeth Y, Durant Y, Gewirtz AT, Sentman CL, Kedl R, Conejo-Garcia JR, 2009b. Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity. J. Clin. Invest 119 (8), 2231–2244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Landry RP, Jacobs VL, Romero-Sandoval EA, DeLeo JA, 2012. Propentofylline, a CNS glial modulator does not decrease pain in post-herpetic neuralgia patients: in vitro evidence for differential responses in human and rodent microglia and macrophages. Exp. Neurol 234 (2), 340–350. [DOI] [PubMed] [Google Scholar]
  30. Oh YK, Suh D, Kim JM, Choi HG, Shin K, Ko JJ, 2002. Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther. 9 (23), 1627–1632. [DOI] [PubMed] [Google Scholar]
  31. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP, 1995. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A 92 (16), 7297–7301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thomas M, Ge Q, Lu JJ, Chen J, Klibanov AM, 2005. Cross-linked small polyethylenimines: while still nontoxic, deliver DNA efficiently to mammalian cells in vitro and in vivo. Pharm. Res 22 (3), 373–380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lisziewicz J, Gabrilovich DI, Varga G, Xu J, Greenberg PD, Arya SK, Bosch M, Behr JP, Lori F, 2001. Induction of potent human immunodeficiency virus type 1-specific T-cell-restricted immunity by genetically modified dendritic cells. J. Virol 75 (16), 7621–7628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ndong C, Landry RP, DeLeo JA, Romero-Sandoval EA, 2012. Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain. Mol. Pain 8, 34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 (4), 402–408. [DOI] [PubMed] [Google Scholar]
  36. Godbey WT, Wu KK, Mikos AG, 1999a. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl. Acad. Sci. U.S.A 96 (9), 5177–5181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jiang HL, Kim YK, Arote R, Jere D, Quan JS, Yu JH, Choi YJ, Nah JW, Cho MH, Cho CS, 2009. Mannosylated chitosan-graft-polyethylenimine as a gene carrier for Raw 264.7 cell targeting. Int. J. Pharm 2, 133–139. [DOI] [PubMed] [Google Scholar]
  38. Martinez-Pomares L, 2012. The mannose receptor. J. Leukoc. Biol 92 (6), 1177–1186. [DOI] [PubMed] [Google Scholar]
  39. Kunath K, von Harpe A, Petersen H, Fischer D, Voigt K, Kissel T, Bickel U, 2002. The structure of PEG-modified poly(ethylene imines) influences biodistribution and pharmacokinetics of their complexes with NF-kappaB decoy in mice. Pharm. Res 19 (6), 810–817. [DOI] [PubMed] [Google Scholar]
  40. Fischer D, Bieber T, Li Y, Elsasser HP, Kissel T, 1999. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res 16 (8), 1273–1279. [DOI] [PubMed] [Google Scholar]
  41. Godbey WT, Wu KK, Mikos AG, 1999b. Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. J. Biomed. Mater. Res 45 (3), 268–275. [DOI] [PubMed] [Google Scholar]
  42. Bonnet ME, Erbacher P, Bolcato-Bellemin AL, 2008. Systemic delivery of DNA or siRNA mediated by linear polyethylenimine (L-PEI) does not induce an inflammatory response. Pharm. Res 25 (12), 2972–2982. [DOI] [PubMed] [Google Scholar]
  43. Mizrahi A, Czerniak A, Levy T, Amiur S, Gallula J, Matouk I, Abu-lail R, Sorin V, Birman T, de Groot N, Hochberg A, Ohana P, 2009. Development of targeted therapy for ovarian cancer mediated by a plasmid expressing diphtheria toxin under the control of H19 regulatory sequences. J. Transl. Med 7, 69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Brunot C, Ponsonnet L, Lagneau C, Farge P, Picart C, Grosgogeat B, 2007. Cytotoxicity of polyethyleneimine (PEI), precursor base layer of polyelectrolyte multilayer films. Biomaterials 28 (4), 632–640. [DOI] [PubMed] [Google Scholar]
  45. Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, Malo D, 1999. Endotoxin-tolerant mice have mutations in toll-like receptor 4 (Tlr4). J. Exp. Med 189 (4), 615–625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B, 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282 (5396), 2085–2088. [DOI] [PubMed] [Google Scholar]
  47. de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE, 1991. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med 174 (5), 1209–1220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Chanput W, Mes J, Vreeburg RA, Savelkoul HF, Wichers HJ, 2010. Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds. Food Funct 1 (3), 254–261. [DOI] [PubMed] [Google Scholar]
  49. Chieppa M, Bianchi G, Doni A, Del Prete A, Sironi M, Laskarin G, Monti P, Piemonti L, Biondi A, Mantovani A, Introna M, Allavena P, 2003. Cross-linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immunosuppressive program. J. Immunol 171 (9), 4552–4560. [DOI] [PubMed] [Google Scholar]

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