A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection

    A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection Olga Scherer, Marten B. Maeß, Saskia Lindner, Ulrike Garscha, Christina Weinigel, Silke Rummler, Oliver Werz, Stefan Lorkowski PII: DOI: Reference:

S0022-1759(15)00121-0 doi: 10.1016/j.jim.2015.04.007 JIM 12012

To appear in:

Journal of Immunological Methods

Received date: Revised date: Accepted date:

27 January 2015 4 April 2015 8 April 2015

Please cite this article as: Scherer, Olga, Maeß, Marten B., Lindner, Saskia, Garscha, Ulrike, Weinigel, Christina, Rummler, Silke, Werz, Oliver, Lorkowski, Stefan, A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection, Journal of Immunological Methods (2015), doi: 10.1016/j.jim.2015.04.007

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ACCEPTED MANUSCRIPT A procedure for efficient non-viral siRNA transfection of

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primary human monocytes using nucleofection

Olga Scherera, Marten B. Maeßb, c, Saskia Lindnera, Ulrike Garschaa, Christina Weinigeld,

University

Jena,

Philosophenweg

[email protected];

14,

07743

Jena,

[email protected];

Germany; E-mail

addresses:

[email protected];

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[email protected] b

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Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller

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a

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Silke Rummlerd, Oliver Werza, Stefan Lorkowskib,*

Chair of Nutritional Biochemistry and Physiology, Institute of Nutrition, Friedrich Schiller

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University Jena, Dornburger Str. 25, 07743 Jena, Germany; E-Mail address: [email protected]; [email protected] Present address: Department of Molecular Imaging, Medical Photonics Research Center,

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c

Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan d

Institute of Transfusion Medicine, University Hospital Jena, Jena, Germany; E-mail

addresses: [email protected]; [email protected]

* Corresponding author: Prof. Dr. Stefan Lorkowski, Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Str. 25, 07743 Jena, Germany; Phone: +49-03641949710; Fax: +49-03641-949712; E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Monocytes are an important constituent of the innate immune system. Therefore,

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manipulating gene expression of primary human monocytes is a crucial mean to study and

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characterize the functions of targeted proteins in monocytes. Gene silencing by transfection of

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cells with small interfering RNA (siRNA) leading to degradation of the corresponding mRNA and thus to reduced target protein levels is an important tool to investigate gene and protein function of interest. However, non-viral transfection of primary monocytes is challenging

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because siRNA uptake by these suspended cells is tricky, the individual cells vary amongst

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different donors, and do not proliferate. Here, we describe a procedure for efficient non-viral transfection of primary human monocytes isolated from peripheral blood, which maintains cell viability and cell functions, such as responsiveness to stimuli like LPS and IL-10.

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Nucleofection was used as an electroporation technique that enables efficient introduction of siRNA and silencing of target genes. Using a modification of our previously published

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protocol for the fast-proliferating THP-1 monocytic cell line, we transfected primary human monocytes with siRNA targeting 5-lipoxygenase (5-LO). In fact, we successfully

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downregulated 5-LO mRNA resulting in reduced protein levels and enzymatic activity.

Key words: Transfection, nucleofection, electroporation, monocytes, siRNA, 5-lipoxygenase.

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ACCEPTED MANUSCRIPT Abbreviations: AA, arachidonic acid; 7-AAD, 7-aminoactinomycin D; ALOX5, arachidonate 5-lipoxygenase

acid;

IL,

interleukin;

5-LO,

5-H(p)ETE, 5-

5-lipoxygenase;

LPS,

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hydroperoxyeicosatetraenoic

FCS, fetal calf serum;

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CD163, cluster of differentiation 163;

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gene; CCL3, chemokine (C-C motif) ligand 3; CCR7, chemokine (C-C motif) receptor 7;

lipopolysaccharide; LTB4, leukotriene B4; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PE, phycoerythrin; PG, prostaglandin; RP-HPLC, reversed-phase

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high-performance liquid chromatography; RPL37A, ribosomal protein L37A; RT-qPCR,

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reverse transcriptase quantitative polymerase chain reaction; siRNA, small interfering RNA;

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SOCS3, suppressor of cytokine signaling 3; TNF, tumor necrosis factor.

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ACCEPTED MANUSCRIPT 1. Introduction As a crucial component of innate immunity, monocytes are recruited from the blood stream

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into inflamed tissues, where they differentiate into macrophages. Monocytes/macrophages

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possess versatile functions in immune defense. They phagocyte pathogens and activate the

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adaptive immunity, thereby protecting the host. Monocytes/macrophages also promote wound healing or angiogenesis and are involved in the pathogenesis of several diseases by releasing proinflammatory mediators that can promote inflammation and even cancer development (De

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Palma, Murdoch et al. 2007, Shi and Pamer 2011). Therefore, monocytes are relevant cells for

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investigation of their cell biology and regulation of cellular responses within immune defense and inflammation. 5-Lipoxygenase (5-LO) plays a pivotal role in inflammatory and allergic conditions and thus in diseases, such as asthma and cardiovascular disease, but also in several

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cancers (Peters-Golden and Henderson 2007). 5-Lipoxygenase initiates the synthesis of leukotrienes (LTs) and other bioactive lipid mediators from arachidonic acid (AA)

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(Samuelsson, Dahlen et al. 1987). Leukotrienes are proinflammatory mediators. For example, LTB4 promotes leukocyte chemotaxis, and cysteinyl LTs, such as LTC4, LTD4 and LTE4,

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cause increased vascular permeability and bronchoconstriction. Among immune cells, including polymorphonuclear leukocytes (PMNL) and mast cells, LTs are formed mainly by monocytes and macrophages. 5-Lipoxygenase is a cytosolic protein, which, upon cell stimulation and intracellular mobilization of Ca2+, translocates to the nuclear membrane. Arachidonic acid is released by the cytosolic phospholipase (cPL)A2 from phospholipids at this locale where it is subjected to 5-LO for metabolism (Radmark, Werz et al. 2007).

Gene silencing approaches require effective uptake of nucleic acids into the cell. Several methods have been published for the introduction of e.g. plasmid DNA or siRNA into eukaryotic cells (Burke, Sumner et al. 2002, Morille, Passirani et al. 2008). However, monocytes are hard to transfect with commonly used methods (Schnoor, Buers et al. 2009). 4

ACCEPTED MANUSCRIPT With our previously published protocol, we successfully established a method, based on electroporation, for the efficient transfection of THP-1 macrophages (Schnoor, Buers et al.

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2009). The THP-1 monocytic leukemia cell line is widely used instead of primary human

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monocytes for gene knockdown experiments and for investigation of the impairment of the

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target protein as consequence for the cellular functionality (Preiss, Namgaladze et al. 2007, Maess, Wittig et al. 2014). One advantage of such immortalized monocytic cell line is their fast-proliferating characteristics that allow convenient culturing and marked de novo protein

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synthesis in contrast to primary monocytes isolated from peripheral blood. However,

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regulation of genes in transformed monocytic cell lines in comparison to non-transformed primary human monocytes may differ (Kohro, Tanaka et al. 2004). Therefore, analysis of gene knockdown in primary monocytes is indispensable to study protein functions in the

respond

to

stimulation

with

interleukin

(IL)-10

or

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Monocytes/macrophages

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context of primary, non-transformed cells.

lipopolysaccharide (LPS) with upregulation of so-called alternative activation marker genes

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(suppressor of cytokine signaling 3 (SOCS3), cluster of differentiation 163 (CD163), IL-10) or classical activation marker genes (IL-1β, tumor necrosis factor (TNF)-α, chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) receptor 7 (CCR7)), according to the M2 (alternative) and M1 (classical) macrophage polarization types (Ito, Ansari et al. 1999, Mantovani, Sozzani et al. 2002). Therefore, responsiveness of monocytes after transfection to either LPS (classical activation stimulus) or IL-10 (alternative activation stimulus) is an important control to verify preservation of characteristics of cellular regulation. Here we present a procedure for the efficient non-viral transfection of primary human monocytes using a modified nucleofection procedure, which maintains cell viability and cell functions, such as responsiveness to LPS and IL-10.

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ACCEPTED MANUSCRIPT 2. Materials and Methods

2.1. Materials

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RPMI 1640 medium with L-glutamine, penicillin, streptomycin, and human serum were from

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PAA Laboratories (Pasching, Austria). Cell culture flasks and well plates were from Greiner

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bio-one (Frickenhausen, Germany). Mouse anti-5-LO monoclonal antibody was a kind gift by Dr. Dieter Steinhilber (Goethe University Frankfurt, Germany). Lipopolysaccharide (LPS)

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and all other chemicals were from Sigma-Aldrich (Steinheim, Germany), unless indicated

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otherwise.

2.2. Isolation of monocytes

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Leukocyte concentrates were prepared from blood of healthy adult human donors who had not

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taken any anti-inflammatory medication for the last 10 days (Institute of Transfusion

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Medicine at the University Hospital Jena, Germany). Peripheral blood mononuclear cells (PBMC) were isolated by dextran sedimentation and centrifugation on lymphocyte separation medium (PAA Laboratories, Pasching, Austria). PBMC were washed with ice-cold phosphate

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buffered saline (PBS) and plated (2  107 cells/ml) in culture flasks containing RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 10% (v/v) fetal calf serum (FCS). After 1.5 hours at 37 °C and 5% CO2, non-adherent cells were removed by washing with PBS. For transfection, adherent monocytes were detached using Accutase I (PAA Laboratories, Pasching, Austria) for 15-30 minutes at 37 °C, 5% CO2 and resuspended in 0.1 % (w/v) glucose in PBS.

2.3. Nucleofection Nucleofection was performed using the Human Monocyte Nucleofector Kit (Lonza, Cologne, Germany) with an adoption of our previously described method (Schnoor, Buers et al. 2009). 6

ACCEPTED MANUSCRIPT For transfection, detached monocytes (2.5  106 cells) were resuspended in 100 µl of Human Monocyte Nucleofector Solution (Lonza, Cologne, Germany) and transferred into a

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nucleofection cuvette. 1 µg siRNA was added and electroporation was performed in the

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Nucleofector 2b device (Lonza, Cologne, Germany) using the Y-001 program. Cells were

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then transferred into a fresh tube containing 500 µl of transfection medium (Lymphocyte Growth Medium (LGM)-3 (Lonza, Cologne, Germany) supplemented with 20% (v/v) human serum, 1% (v/v) non-essential amino acids, 2 mM L-glutamine, 1% (v/v) sodium pyruvate).

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Transfected cells in suspension were then transferred dropwise into two wells of a 12 well-

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plate containing 1.75 ml pre-warmed transfection medium. Within our previously published original protocol, the Amaxa Human Monocyte Nucleofector Medium (Lonza, Cologne,

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Germany) was used. Because of the unavailability of that culture medium, we accordingly

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confirmed suitability and used the LGM-3 medium. After 4 h, medium was removed, replaced by fresh transfection medium (see above, additionally containing 100 U/ml penicillin and 100

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µg/ml streptomycin) and cells were incubated for the indicated times. 24 h after transfection, fresh transfection medium (containing 10% (v/v) human serum) was added and cells were

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cultured as indicated in the figures. The following siRNAs were used: on-target plus human ALOX5 siRNA SMARTpool, on-target plus non-targeting siRNA (Thermo Scientific Dharmacon, Lafayette, CO), Alexa 488 siRNA (Qiagen, Hilden, Germany).

2.4. Flow cytometry analysis For flow cytometry analysis, cells were detached using Accutase I solution (PAA Laboratories, Pasching, Austria) for 15-30 min at 37 °C and 5% CO2. After detachment, cells were centrifuged, resuspended and analyzed with a Attune Acoustic Focusing Cytometer (Life Technologies, Carlsbad, CA). For determination of cell viability and analysis of apoptosis or necrosis, cells were stained with phycoerythrin (PE)-labeled Annexin V and 7aminoactinomycin D (7-AAD), respectively, using the Apoptosis Detection Kit I (BD 7

ACCEPTED MANUSCRIPT Biosciences, Heidelberg, Germany) according to the manufacturer´s instructions. For determination of transfection efficiency, Alexa Fluor 488 labeled non-targeting control siRNA

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(Qiagen, Hilden, Germany) was used.

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2.5. Life cell imaging

For imaging of fluorescently labeled Alexa Fluor 488 siRNA uptake, monocytes (1.25  106 per dish) were plated after nucleofection with fluorescent siRNA into glass bottom dishes

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(MatTek Corporation, MA) containing pre-warmed transfection medium and incubated at 37

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°C and 5% CO2 atmosphere for 24 h. For microscopic imaging, monocytes were washed with pre-warmed PBS and imaging buffer (PBS containing 0.1% (w/v) glucose, 1 mM CaCl 2 and 1

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mM MgCl2) was added. Images were taken with an AxioCam MR3 camera (Carl Zeiss, Jena,

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Germany) and were acquired, cut, linearly adjusted in the overall brightness and contrast, and exported to TIF format using AxioVision 4.8 software. The microscope incubator (Axio

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Observer Z1 inverted microscope, LCl Plan-Neofluar 63x/1.3 Imm Corr DIC M27 objective,

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Carl Zeiss, Jena, Germany) was used at 37 °C and 5% CO2.

2.6. Quantitative real-time RT-PCR (RT-qPCR) After transfection and incubation of monocytes, culture medium was removed and total RNA from monocytes was isolated using the Nucleospin RNA XS Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer´s instructions. For cDNA synthesis, the Superscript III First-Strand Synthesis Supermix was used (Invitrogen, Life Technologies, Carlsbad, CA) with the Flex Cycler2 device (Biometra, Göttingen, Germany). Each sample contained cDNA converted from 33 to 117 ng total RNA. The cDNA was quantified in duplicates using the Maxima SYBR Green qPCR master mix (Thermo Fisher Scientific, MA) and 200 nM of forward and reverse primer on the Lightcycler 480 Real-time PCR detection system (Roche, Mannheim, Germany). Thermal cycling conditions were: initial denaturation at 95 °C for 10 8

ACCEPTED MANUSCRIPT min, then 40 cycles of 15 sec at 95 °C and 60 sec at 60 °C. To calculate the relative mRNA expression the 2(ΔΔC(T)) method was used (Livak and Schmittgen 2001). PCR products were

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size-validated by agarose gel electrophoresis. Primers targeting the genes ALOX5 and ACTB

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were from TIB Molbiol (Berlin, Germany) and primers of the genes CCL3, CCR7, CD163,

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IL1B, IL10, RPL37A, SOCS3 and TNFA were from Life Technologies (Darmstadt,

2.7. SDS-PAGE and Western blot analysis

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Germany).

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After 24, 48 or 72 h of monocyte transfection with siRNA, plates were placed on ice, and culture medium was removed. Then, 50 µl of NP-40 lysis buffer (1% (v/v) NP-40, 1 mM

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Na3VO4, 5 mM EDTA, 10 mM NaF, 5 mM sodium pyrophosphate, 25 mM β-

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glycerophosphate with freshly added 60 µg/ml trypsin inhibitor from soybean, 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin hemisulfate salt in TBS, pH 7.4) were

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added per well and cell culture plates were shaken at 4 °C for 5 min. Monocytes were scraped, transferred into fresh tubes and vortexed intensely. The samples were placed on ice

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for 30 minutes with occasional vortexing and centrifuged at 10,000  g and 4 °C for 5 min. After addition of 4  SDS loading buffer (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 1% (v/v) β-mercaptoethanol, 12.5 mM EDTA, 0.02% (w/v) bromphenol blue), samples were denatured for 5 min at 96 °C. Equal volumes per sample were used. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Biosciences, Little Chalfont, UK). After blocking with 5% (w/v) BSA in TBS plus 0.1% (w/v) Tween-20 for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C. Antibodies against 5-LO (monoclonal from mouse, diluted 1:8, Dr. Steinhilber, Goethe University Frankfurt, Germany) and β-actin (polyclonal from rabbit, diluted 1:1000, Cell Signaling Technology, MA, USA) were used. Membranes were washed 9

ACCEPTED MANUSCRIPT and incubated with fluorescently-labeled secondary antibodies for 40 min at room temperature in the dark. The secondary antibodies IRDye 680LT conjugated goat polyclonal anti-mouse or

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anti-rabbit IgG (1:80,000) and IRDye 800CW conjugated goat polyclonal anti-mouse or anti-

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rabbit IgG (1:10,000) (LI-COR Biosciences, Bad Homburg, Germany) were used. After

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washing the membranes, proteins were detected and band intensities were quantified (after background correction) using the Odyssey imaging system and the Odyssey application

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software 3.0.25 (LI-COR Biosciences, Bad Homburg, Germany).

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2.8. Determination of 5-LO product synthesis

Monocytes were stimulated and 5-LO products were analyzed as described before (Pergola, Rogge et al. 2011). In brief, growth medium was replaced by PGC buffer (PBS containing

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0.1% (w/v) glucose and 1 mM CaCl2) and adherent monocytes were stimulated by addition of 2.5 µM Ca2+-ionophore A23187 and 10 µM arachidonic acid for 10 min at 37 °C. The

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reaction was stopped on ice, methanol was added and 5-LO metabolites in the supernatant were purified and quantified as follows. Acidified PBS and prostaglandin (PG) B1 as an

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internal standard were added to the supernatant and subjected to solid phase extraction on C18-columns (100 mg, UCT, Bristol, PA, USA). Metabolites of 5-LO were eluted by methanol and analyzed by RP-HPLC using C-18 Radial-PAK column (Waters, Eschborn, Germany), as described before (Werz, Szellas et al. 2002). Products of 5-LO include the alltrans-isomers of LTB4, LTB4, and 5-H(p)ETE.

2.9. Stimulation of monocytes To assure that transfection of siRNA does not alter monocyte functionality in general, we analyzed the susceptibility of the cells towards various stimuli. For determination of the responsiveness of primary human monocytes to either LPS or IL-10, cells were transfected with non-targeting siRNA as described above. After 24 h, the cell culture medium was 10

ACCEPTED MANUSCRIPT removed and transfection medium (without serum) was added. Then, cells were stimulated with recombinant human IL-10 (BD Pharmingen, Heidelberg, Germany) (50 ng/ml), LPS

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from E. coli (10 ng/ml) or left unstimulated for 24 h at 37 °C and 5% CO2. Cells were lysed

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and RT-qPCR was performed as described above. Relative mRNA expression of classical

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activation markers (IL-1β, TNF-α, CCL3, CCR7) or alternative activation markers (SOCS3, CD163, IL10) was quantified using RPL37A as reference gene for normalization. In the supernatants of the LPS-stimulated cells, PGE2 formation was determined using a PGE2

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ELISA Kit (Cusabio, Wuhan, China) according to the manufacturer´s instructions.

2.10. Statistics

The data are presented as mean + SEM of n experiments. Statistical analysis was performed

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by Student t test. Data were analyzed with GraphPad InStat program (GraphPad Software,

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CA). A P value of < 0.05 was considered significant.

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ACCEPTED MANUSCRIPT 3. Results and discussion

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We here describe the efficient transfection of freshly isolated primary human monocytes with

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siRNA using nucleofection and present a systematic evaluation of the functionality and

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viability of the transfected cells.

We developed a nucleofection protocol which has been optimized from a previously

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published procedure for the transfection of THP-1 macrophages (Schnoor, Buers et al. 2009).

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Instead of the Amaxa Human Monocyte Nucleofector Medium (Lonza, Cologne, Germany), which has been used in our original transfection protocol, we evaluated the LGM-3 medium from Lonza (Cologne, Germany) for monocyte cultivation after nucleofection. The cell

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culture medium used after nucleofection is essential for remaining high cell viability and functionality, as discussed previously (Maess, Wittig et al. 2014). In our experiments,

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cultivation of transfected monocytes in the LGM-3 led to high viability rates with very low levels of apoptotic (1.86%) or necrotic cells (0.49%) after 24 h (Fig 1A), which were not

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significantly different from that of cells prior to transfection (1.36% apoptotic and 0.20% necrotic cells, Fig 1A). Moreover, we used 20% human serum for the transfection medium, which improved maintenance of monocyte viability. After 24 h, we reduced the human serum to 10% without loss of viability. Particularly, transfected monocytes did not show morphological signs of cell death, such as bleb formation, shrinkage or fragmentation (data not shown).

A critical point of the transfection procedure is the detachment of monocytes. Detachment of primary monocytes was reliably achieved with Accutase I detachment solution. However, small amounts of cell debris could not be avoided upon detachment (Fig 1A), but were independent of the transfection (data not shown). 12

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In order to confirm the uptake of siRNA into monocytes, we transfected cells with Alexa

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Fluor 488 labeled control siRNA. Flow cytometric analysis showed transfection efficiencies

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of almost 100%, as the whole population of transfected cells showed a shift in the histogram

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plot, verifying an increase in intracellular fluorescence (Fig. 1B). Furthermore, fluorescent siRNA in transfected monocytes was visualized using life cell imaging in order to confirm flow cytometric results. Our data show a consistent and uniform intracellular fluorescence

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distribution 24 h after transfection (Fig. 1B). This is in contrast to chemical approaches used

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for the transfection of adherent monocytes/macrophages that often result, for example in case of XtremeGene siRNA, in good cell morphology and cell vitality but inhomogeneous siRNA distribution within THP-1 macrophages. Fluorescence imaging analyses indicate that siRNA

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molecules after transfection with XtremeGene siRNA are not homogeneously distributed within the cytosol like after nucleofection. They rather form bright agglomerates within the

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cells (Supplementary Fig. S1). Therefore, we consider nucleofection the superior approach as the resulting population of transfected cells is considerably more homogeneous

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(Supplementary Fig. S1) and the intracellular distribution of the siRNA molecules is more homogeneous as well.

Another advantage of the nucleofection approach is the avoidance of lipid-based reagents. Those are known to remain within the cells, often affecting cell viability and causing offtarget effects (Filion and Phillips 1997). Furthermore, lipid-siRNA complexes were shown to exhibit immunostimulatory effects likely through endosomal toll-like receptor signaling pathways (Judge, Sood et al. 2005). Next, lipid-delivered siRNA enters endosomes which mature to lysosomes, thus requiring release of the siRNA into the cytosol (Simoes, Moreira et al. 2004). This is often achieved by lysis of lysosomes and consequently means that the cytosol is exposed to lysosomal enzymes which may cause adverse cellular reactions. 13

ACCEPTED MANUSCRIPT However, meanwhile, also successful lipid-based gene knockdown protocols for primary monocytes have been published (Troegeler, Lastrucci et al. 2014). A major limitation of

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nucleofection is the expensive equipment and the necessity to detach cells for efficient

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nucleofection, making this technique time-consuming and unsuitable for some cell types. By

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contrast, lipid-based reagents are cheap, fast and are suitable for high-throughput experiments without detaching the cells of interest. Despite these limitations, nucleofection is a reliable and efficient transfection method allowing homogeneous transfection as well as cellular

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distribution.

Next, we aimed to proof successful transfection of siRNA by analyzing the functional biological outcome. We transfected primary human monocytes with siRNA targeting the

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mRNA of the 5-LO gene (ALOX5), which is mainly expressed in monocytes/macrophages and neutrophils (Radmark, Werz et al. 2007). In resting leukocytes, 5-LO is a soluble protein

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that initiates formation of leukotrienes from arachidonic acid (AA) (Radmark, Werz et al. 2007) and is linked to various allergic and inflammatory diseases (Peters-Golden and

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Henderson 2007), where monocytes and macrophages play major roles. Real-time RT-PCR experiments reveal that in comparison to non-targeting control siRNA, the transfection of ALOX5 siRNA reduced mRNA expression by 56% after 24 h, by 60% after 48 h and by 57% after 72 h, respectively (Fig. 2A). In a previous study, we found mRNA reduction rates of about 75% using an nucleofection approach optimized for the THP-1 macrophage model (Schnoor, Buers et al. 2009), implying that ALOX5 siRNA targeted knockdown might be further improved, which will be addressed in future studies. However, the different knockdown efficiencies might be explained by the type of applied siRNA or differences in the chosen target mRNA, for example due to different mRNA stabilities.

We next investigated if 5-LO protein levels were reduced after transfection of ALOX5 siRNA 14

ACCEPTED MANUSCRIPT as a consequence of the downregulation of 5-LO mRNA. Western blot analyses showed that cells transfected with ALOX5-specific siRNA exhibited a clear reduction of 5-LO protein

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levels by 25% after 24 h, by 24% after 48 h and by 53% after 72 h of transfection compared to

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cells transfected with non-targeting control siRNA (Fig. 2B). In agreement with the literature,

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5-LO protein levels continuously declined per se during incubation of monocytes, visible at 24 h up to 72 h (Fig. 2B). Previous studies by our group and others (Ring, Riddick et al. 1996, Wagner, Werz et al. 2013) have shown that simple cultivation of monocytes under standard

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culture conditions downregulates 5-LO expression and 5-LO (and FLAP) protein levels

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within 6 h up to 7 days. The cultivation of the transfected monocytes in our experimental setup may therefore counteract the siRNA-mediated knockdown by general decrease of 5-LO

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mRNA and protein expression.

The downregulation of 5-LO protein due to ALOX5 siRNA in comparison to non-targeting

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siRNA was confirmed by investigation of 5-LO enzyme activity. The capacity of 5-LO to produce AA metabolites was analyzed in the transfected monocytes after challenge with the

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Ca2+ ionophore A23187 in combination with AA, as previously described (Pergola, Rogge et al. 2011). 5-Lipoxygenase product formation in monocytes transfected with ALOX5-specific siRNA was slightly reduced after 24 h (by 19%) and significantly diminished after 48 h (by 26%) versus cells that were treated with non-targeting control siRNA (Fig. 2C). For monocytes that were analyzed 72 h after transfection, the 5-LO product levels, likely due to decreasing 5-LO expression during monocyte cultivation, were under the detection limit and could not be clearly assessed. Interestingly, although downregulation of 5-LO protein 48 h after ALOX5 siRNA transfection was slightly less pronounced than after 24 h (Fig. 2B), suppression of 5-LO product formation (by 26%) was significant only after 48 h (Fig. 2C). However, previous studies have shown that cellular 5-LO activity does not necessarily correlate with the amount of 5-LO protein (Jakobsson, Steinhilber et al. 1992, Werz and 15

ACCEPTED MANUSCRIPT Steinhilber 1996).

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Taken together, downregulation of 5-LO mRNA led to reduced 5-LO protein expression and

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subsequently to a decrease of the measureable 5-LO enzyme activity. In contrast to many

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other cell types, the rate of proliferation of primary monocytes in vitro as well as in vivo is quite low (Treves 1985); therefore the rate of protein de novo synthesis in general and in case of 5-LO is low (Ring, Riddick et al. 1996, Wagner, Werz et al. 2013). Together with the fact

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that 5-LO expression declines during cultivation, this may hamper 5-LO mRNA knockdown.

To assure that the process of nucleofection per se did not alter monocyte functionality, we analyzed mRNA expression of distinct marker genes after stimulation with the classical

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activating stimulus LPS (M1 polarization) and after stimulation with the alternative activation stimulus IL-10 (M2 polarization) in monocytes transfected with non-targeting control siRNA.

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As M1 marker genes of the classical activation program we analyzed IL1B, TNFA, CCL3, CCR7 as well as SOCS3, CD163 and IL10 as M2 markers for the alternative stimulation

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pathway. As it has been reported for non-transfected naïve monocytes/macrophages (Ito, Ansari et al. 1999, Stoiber, Kovarik et al. 1999, Mantovani, Sozzani et al. 2002), expression of the tested M1 marker genes IL1B, TNFA, CCL3, CCR7 plus the M2 marker genes SOCS3 and IL10 was significantly increased by stimulation with LPS (Fig. 3A), whereas stimulation with IL-10 led to significantly increased expression of SOCS3, CD163 and IL10 (Fig 3B). Responses of monocytes to different stimuli are diverse and thus upregulation of distinct mRNA expression is not exclusively restricted to a certain stimulus. For example, previous studies have shown that induction of SOCS-3 mRNA expression is measureable after stimulation with both, LPS (Stoiber, Kovarik et al. 1999) and IL-10 (Ito, Ansari et al. 1999). Our data also show that particularly the M2 marker genes SOCS-3 and IL-10 are significantly activated by IL-10 and LPS, respectively (Fig. 3B). A further characteristic feature of 16

ACCEPTED MANUSCRIPT monocytes is the secretion of PGE2, a lipid mediator involved in fever, inflammation and pain, in response to LPS (Kurland and Bockman 1978). To further confirm functionality of

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transfected monocytes, we therefore measured PGE2 levels in supernatants of the transfected

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cells stimulated with LPS. Our data show that the transfected monocytes strongly respond to

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LPS releasing PGE2 (Fig. 3C). Therefore, following our transfection protocol, monocytes are generally still susceptible to different stimuli and do characteristically respond to either the

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classical stimulus LPS or the alternative stimulus IL-10.

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Taken together, our study presents an easy to follow nucleofection-based protocol for the efficient transfection of primary human monocytes with siRNA without loss of cell viability or cell functionality. We evaluated the protocol by successfully targeting the 5-LO, a soluble

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protein mainly expressed in monocytes/macrophages and neutrophils with pivotal roles in inflammation and allergy. Downregulation of 5-LO in monocytes was confirmed by reduced

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mRNA and protein levels, as well as diminished cellular 5-LO product synthesis. This

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protocol can be easily transferred to other target proteins of primary human monocytes.

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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (DFG) FOR 1406,

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WE2260/11-1, SFB 1127 ChemBioSys, SFB/TR 124 FungiNet, GRK 1715 as well as the

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Conflict of Interest Statement: None declared.

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Ernest-Solvay-Stiftung and the Thüringer Ministerium für Bildung, Wissenschaft und Kultur.

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ACCEPTED MANUSCRIPT Figure legends

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Figure 1. Cell population, cell viability and transfection efficiency of primary human

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monocytes. (A) Cell population of monocytes (upper plots) prior to nucleofection (left) and

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24 h after nucleofection (right). Cell viability (middle and lower plots) before (left) and 24 h after (right) transfection of siRNA. Flow cytometric analysis of PE Annexin V and 7-aminoactinomycin D (7-AAD) staining was performed for the detection of apoptotic and necrotic

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cells, respectively. Plots are representatives out of three independent experiments. (B)

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Transfection efficiency was determined by flow cytometric analysis after transfection of Alexa Fluor 488 labeled siRNA which revealed an efficiency of almost 100%. Transfection was also verified by imaging of monocytes after uptake of fluorescent-labeled Alexa 488

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siRNA (green). Scale bar: 10 µm. Images are representatives out of two independent

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experiments.

Figure 2. Analysis of 5-LO in transfected monocytes. (A) Quantitative real-time RT-PCR

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analysis of ALOX5 mRNA in transfected monocytes. Transfection with ALOX5-directed siRNA resulted in a significant downregulation of ALOX5 mRNA expression in comparison to transfection of non-targeting siRNA as control. Monocytes (1.25 106) were transfected with siRNA using the Nucleofector approach and mRNA expression was analyzed after indicated time points. Normalization to the reference gene ACTB was performed. Data are means + SEM of fold mRNA expression; n =3-4. (B) 5-Lipoyxgenase protein analyses of transfected monocytes. Western blot analysis showed downregulated 5-LO protein after transfection of monocytes with ALOX5 siRNA in comparison to cells transfected with nontargeting siRNA. Monocytes were pelleted, lysed and equal sample volumes were analyzed. Data from densitometric analysis are means + SEM of fold 5-LO/β-actin ratio. A 23

ACCEPTED MANUSCRIPT representative Western blot of 3-4 independent experiments is shown. (C) 5-LO activity detection in transfected monocytes. Monocytes at indicated time points after transfection with

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either ALOX5 or non-targeting control siRNA were stimulated with 2.5 µM A23187 and 10

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µM AA for 10 min at 37 °C. Supernatants were then removed and analyzed by HPLC

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following solid phase extraction and elution by methanol. 5-Lipoxygenase products: all transisomers of LTB4, LTB4 and 5-H(p)ETE. Data are means + SEM; n =3. 100% control (nontargeting siRNA) after 24 h: 34.66 ± 3.59 ng. 100% control (non-targeting siRNA) after 48 h:

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9.75 ± 4.64 ng. After 72 h, 5-LO product formation was not detectable (n.d.).

Figure 3. Responsiveness of monocytes to LPS and IL-10 after nucleofection. Primary

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human monocytes were transfected with non-targeting siRNA and plated (1.25  106/well)

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into 12-well plates. 24 h after transfection cells were stimulated with IL-10 (50 ng/ml), LPS (10 ng/ml) or left untreated. After further 24 h, RNA was isolated and relative mRNA

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expression of (A) M1 markers (IL1B, TNFA, CCL3, CCR7) or (B) M2 markers (SOCS3, CD163, IL10) was determined using RT-qPCR. Normalization to the reference gene RPL37A was performed. Data are expressed as mean + SEM, n = 3. (C) In the supernatants of LPSstimulated monocytes, PGE2-levels were determined by ELISA. Data are expressed as mean + SEM, n = 3.

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ACCEPTED MANUSCRIPT Table 1 Overview of PCR primer sequences used in this study. Abbreviatio GenBank n Acc. no.

Size of PCR Forward primer produc t (bp)

Reverse primer

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Target gene

NM_000698. 3

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5’-ACTGGCTGAATGACGACTGG 5’-CAGGGGAACTCGATGTAGTCC

β-actin

ACTB

NM_001101. 3

125

5‘-CTCACCGAGCGCGGCTACGA 5‘-GGAGCTGGAAGCAGCCGTGG

CCL3

NM_002983. 2

82

5‘ CTGACTACTTTGAGACGAGCAG 5‘-TCAGCACAGACCTGCCGG CC

CCR7

NM_001838. 3

89

5‘AATGAAAAGCGTGCTGGTGGT

99

5‘-TCTGTTGGCCATTTTCGTCG

5‘TGGTGGACTAAGTTCTCTCCTCTT GA

115

5‘-TCCCTGCCCACAGACCTTC

5‘-GTGCATCGTGCACATAAGCCT

NM_000572. 2

88

RPL37A

NM_000998. 4

94

SOCS3

NM_003955. 3

TNFA

NM_000594. 1

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5‘TCACTCATGGCTTTGTAGATGCC

5‘-ATTGAAATCAGCCAGCACGC

5‘-AGGAACCACAGTGCCAGATCC

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Ribosomal protein L37A Suppressor of cytokine signaling 3 Tumor necrosis factor-α

IL10

5‘CCTGTGAAAACAAGAGCAAGG C

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159

5‘GGATTCTCCTTCAATTCCTCAG CT

5‘CTGGCAGTTCTCATTAGTTCAGC A

5‘TCTTCTCGAACCCCGAGTGAC

5‘-GGTACAGGCCCTCTGATGGC

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Interleukin10

IL1B

5‘-TGTCTCCGATGTAATCGTCCGT

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Interleukin1β

NM_203416. 2; NM_004244. 4 NM_000576. 2

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Cluster of differentiatio CD163 n 163

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Chemokine (C-C motif) ligand 3 Chemokine (C-C motif) receptor 7

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Arachidonat e 5ALOX5 lipoxygenas e gene

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ACCEPTED MANUSCRIPT Highlights

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We describe an efficient method for siRNA transfection of primary human monocytes based on nucleofection. siRNA targeting 5-lipoxygenase (5-LO) efficiently downregulated 5-LO mRNA resulting in reduced 5-LO protein levels and enzymatic activity. Our method maintains viability of transfected monocytes and their responsiveness to common stimuli.

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