Elicit: Mechanism of Adalimumab in TNF-alpha Inhibition

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Mechanism of Adalimumab in TNF-alpha Inhibition

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May 5, 2026

TNF-alpha adalimumab mechanism

Adalimumab neutralizes TNF-alpha through high-affinity binding that blocks receptor interaction, while simultaneously inducing apoptosis in transmembrane TNF-alpha-expressing cells via complement-dependent and caspase-mediated mechanisms and modulating intracellular inflammatory pathways including NF-κB signaling and microRNA networks.

Abstract

Adalimumab functions through five complementary mechanisms to inhibit TNF-alpha activity. Structurally, adalimumab binds TNF-alpha with high affinity (KD 7.05-10 × 10⁻¹¹ M) through a large epitope (2,540 Ų buried surface area) that directly occupies the TNF receptor-binding site, blocking both soluble and transmembrane TNF-alpha. Beyond neutralization, adalimumab induces cytotoxic effects including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and caspase-3-mediated apoptosis in transmembrane TNF-alpha-expressing cells. Adalimumab modulates intracellular signaling by reversing NF-κB pathway activation and downregulating genes including IKBKB, IRAK1, TRAF2, and MAP3K7, while simultaneously regulating anti-inflammatory microRNAs (miR-1297, miR-30a, miR-126-3p) and pro-inflammatory microRNAs (miR-146a-5p). Cell-type-specific effects include restoration of intracellular TNF-alpha levels in monocytes, CD36 upregulation through NADPH oxidase-mediated redox signaling, suppression of endothelial adhesion molecules, and TNF-RI blockade in osteoclasts.

Compared to other anti-TNF agents, adalimumab demonstrates a larger antigen-antibody interface than infliximab and unique complex-forming capacity unlike the receptor fusion protein etanercept. Adalimumab and infliximab both induce outside-to-inside reverse signaling and cell cycle arrest, mechanisms absent with etanercept that may explain their superior efficacy in Crohn’s disease and Wegener’s granulomatosis. Clinical applications are supported by biomarker potential (intracellular TNF-alpha levels, NF-κB-associated microRNAs), though neutralizing anti-adalimumab antibodies represent a resistance mechanism. The temporal hierarchy shows immediate binding effects, gene expression changes within 2-8 hours, and phenotypic restoration over months, indicating acute anti-inflammatory activity transitions to sustained cellular reprogramming.

Methods

We analyzed 10 sources from an initial pool of 200, using 8 screening criteria. Each paper was reviewed for 6 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Mechanistic Focus, Mechanistic Measurements, Study Design, Adalimumab Population, Mechanistic Investigation, Adalimumab-Specific Data, Mechanistic Insights, Publication Type

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “TNF-alpha adalimumab mechanism”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Identify the primary mechanism of adalimumab action being studied, categorizing as:

Extract details about the experimental system used to study adalimumab mechanisms, including:

Extract the main discoveries about how adalimumab works at the molecular/cellular level, including:

Identify which specific cell types, tissues, or biological systems adalimumab mechanisms were studied in, such as:

Extract any comparisons made between adalimumab and other anti-TNF agents (infliximab, etanercept, certolizumab, golimumab) regarding mechanisms of action, including:

Extract information connecting the mechanistic findings to clinical applications or disease treatment, including:

Results

Characteristics of Included Studies

Study

Full Text Retrieved?

Experimental Model

Mechanism Category

Disease Context

Aleksandra Plata-Babula et al., 2025

Yes

HaCaT keratinocytes (in vitro)

Binding/neutralization, Cellular regulation

Psoriasis

Shi Hu et al., 2013

Yes

Crystal structure, E. coli and CHO cells (in vitro)

Binding/neutralization (epitope binding, receptor blockade)

TNF-alpha-associated diseases

Carlos Zamora-Atenza et al., 2014

Yes

Primary monocytes from RA patients and healthy controls (in vitro and human)

Binding/neutralization, Cytotoxic effects, Signal transduction, Cellular regulation, Tissue/organ effects

Rheumatoid arthritis

Raghav Oberoi et al., 2016

Yes

THP-1 macrophages, HUVECs, hypercholesterolemic mice (in vitro and in vivo)

Binding/neutralization, Tissue/organ effects

Atherosclerosis, rheumatoid arthritis

H. Mitoma et al., 2008

No

Jurkat T cells (in vitro)

Binding/neutralization, Cytotoxic effects, Signal transduction

Rheumatoid arthritis and inflammatory disorders

J. Boyer et al., 2007

Yes

Primary human monocytes (in vitro)

Signal transduction, Binding/neutralization

Chronic inflammatory diseases, rheumatoid arthritis

Shi Hu et al., 2014

Yes

Structural studies (crystallography)

Binding/neutralization (epitope binding, receptor blockade)

TNF-alpha-associated diseases

Chong Shen et al., 2006

No

THP-1 cells, SCID-Beige mice (in vitro and in vivo)

Binding/neutralization, Cytotoxic effects

Crohn’s disease and rheumatoid arthritis

B. Harvey & Z. Kaymakcalan, 2014

No

Primary human osteoclast precursors (in vitro)

Binding/neutralization (receptor blockade)

TNF-alpha-related conditions

F. Prattichizzo et al., 2016

Yes

HUVECs, MCF-7 cells, CACs from psoriasis patients (in vitro and human)

Binding/neutralization, Cellular regulation, Tissue/organ effects

Psoriasis, endothelial senescence

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The included studies employed diverse experimental systems ranging from structural crystallography to primary human cells and animal models. Four studies had full-text availability limitations but provided mechanistic insights through their abstracts. The studies investigated adalimumab mechanisms across multiple cell types including keratinocytes, monocytes/macrophages, T cells, endothelial cells, and osteoclasts, reflecting the broad cellular targets of TNF-alpha signaling.

Thematic Analysis of Adalimumab Mechanisms

Molecular Binding and TNF-Alpha Sequestration

Structural studies revealed the precise molecular basis of adalimumab’s interaction with TNF-alpha. Adalimumab binds through a large, highly complementary interface with a buried surface area of 2,540 Ų. The epitope comprises discontinuous segments including residues TNFPro-20, TNFGln-21, TNFGlu-23, TNFLys-65 to TNFGln-67, TNFGlu-110 to TNFPro-113, TNFTyr-141, TNFAla-145 to TNFGlu-146, and TNFThr-72, TNFHis-73, TNFThr-77, TNFThr-79, TNFSer-81, TNFLys-90 to TNFAsn-92, and TNFGlu-135 to TNFAsn-137 of an adjacent TNF-alpha protomer. This binding directly occupies the TNF receptor-binding site, preventing TNF-alpha from interacting with its receptors.

The affinity of adalimumab for TNF-alpha is high, with KD values ranging from 7.05 × 10⁻¹¹ M to 1.0 × 10⁻¹⁰ M, superior to both etanercept and infliximab. Adalimumab binds both soluble and transmembrane TNF-alpha, enabling it to block TNF-alpha signaling through multiple mechanisms. The formation of adalimumab-TNF complexes appears critical to its function, with cross-linking experiments demonstrating that complex formation enhances inhibitory effects two-fold.

Cytotoxic Mechanisms

Adalimumab induces apoptosis in transmembrane TNF-alpha-expressing cells through multiple pathways. In T cells expressing transmembrane TNF-alpha, adalimumab and infliximab demonstrated nearly equal complement-dependent cytotoxicity (CDC) activities, substantially higher than etanercept. Antibody-dependent cell-mediated cytotoxicity (ADCC) activities were similar among all three anti-TNF agents.

Beyond CDC and ADCC, adalimumab induces apoptosis through activation of intracellular caspases. Specifically, caspase-3 activation was demonstrated following adalimumab treatment, and this apoptotic effect could be abrogated by pan-caspase inhibitors in vivo. Adalimumab also induced cell cycle arrest in transmembrane TNF-alpha-expressing cells, reflecting outside-to-inside signal transduction through transmembrane TNF-alpha.

Signal Transduction and Pathway Modulation

NF-κB Pathway Regulation

In keratinocytes stimulated with lipopolysaccharide, adalimumab reversed the upregulation of multiple NF-κB-associated genes including IKBKB, IRAK1, TRAF2, MAP3K7, and TNFAIP3, with concordant changes at the protein level. STRING analysis identified IKBKB as a central hub in the protein-protein interaction network. The effects were observed across multiple timepoints (2, 8, and 24 hours), with adalimumab consistently downregulating pro-inflammatory transcripts and inhibiting NF-κB-dependent transcription.

MAPK Pathway Effects

Adalimumab reduced concentrations of proteins involved in MAPK pathways. Specific protein targets included TNFAIP3, BCL2L1, CXCL2, and MAP3K7, demonstrating coordinated regulation across inflammatory signaling networks.

CD36 and Lipid Metabolism

In human monocytes, adalimumab increased both CD36 membrane expression and mRNA levels by 59% and 90% in the presence and absence of TNF-alpha, respectively. This effect occurred through redox signaling via NADPH oxidase activation, with a two-fold increase in reactive oxygen species production. The time course showed no effect at 6 or 12 hours, but a 92% increase by 24 hours. Importantly, this mechanism was independent of the Fc portion of adalimumab, as the F(ab’)2 fragment was responsible for the increase in CD36 expression.

MicroRNA Modulation

Adalimumab regulated multiple microRNAs with anti-inflammatory and pro-apoptotic functions. In keratinocytes, several regulatory miRNAs including miR-1297, miR-30a, miR-95-5p, miR-125b, and miR-4329 showed reciprocal expression changes consistent with anti-inflammatory activity. In endothelial cells and circulating angiogenic cells, adalimumab significantly reduced miR-146a-5p expression and increased miR-126-3p expression. The reduction in miR-146a-5p correlated with decreased expression of its target Irak1, while increased miR-126-3p was associated with decreased Spred1 expression.

Cellular Regulation and Phenotype Modulation

Monocyte Regulation

In rheumatoid arthritis patients, intracellular TNF-alpha (iTNF-alpha) in CD14+ monocytes was significantly lower (33.16 ± 4.82%) than in healthy donors (66.51 ± 2.4%). After adalimumab treatment, there was a progressive increase in iTNF-alpha+ CD14+ cells over 12 injections, with levels becoming comparable to healthy donors in patients with good to moderate EULAR-defined responses. This effect was accompanied by reductions in inflammatory cytokines (IL-1, IL-6, IL-8) and acute-phase reactants (CRP, fibrinogen, ESR).

Endothelial Function

Adalimumab prevented endothelial activation by suppressing mRNA and protein expression of adhesion molecules including VCAM-1, ICAM-1, and E-selectin within 3 to 6 hours. This led to reduced monocyte adhesion under both static and flow conditions and suppressed endothelial leakage. In senescent endothelial cells, adalimumab reduced IL-6 release and increased eNOS expression, contributing to improved endothelial barrier function.

Osteoclast Inhibition

In osteoclast precursors, adalimumab inhibited TNF-enhanced osteoclast function more effectively than etanercept, with IC50 values of 9.6 nM for adalimumab versus >130 nM for etanercept. The mechanism involved restricting TNF access to TNF-RI, and the Fc domain did not contribute to these inhibitory effects, as the F(ab’)2 fragment showed equal efficacy.

Comparative Mechanisms Among Anti-TNF Agents

Four studies directly compared adalimumab with other anti-TNF biologics, revealing both shared and unique mechanistic properties.

Structural Differences

Compared to infliximab, adalimumab demonstrated a larger antigen-antibody interface, with more extensive overlap with the TNF-alpha-TNFR2 interface. This structural difference translated to higher affinity for TNF-alpha and more predictable clinical effects. Unlike etanercept, which is a TNF receptor:Fc fusion protein, adalimumab forms stable complexes with TNF-alpha through its antibody structure.

Cytotoxic Mechanisms

While all three agents bound to transmembrane TNF-alpha, adalimumab and infliximab showed similar CDC activities that were considerably higher than etanercept. ADCC activities were nearly equal among the three agents. Uniquely, adalimumab and infliximab induced apoptosis and cell cycle arrest through outside-to-inside signal transduction, which may explain their superior efficacy in Crohn’s disease and Wegener’s granulomatosis compared to etanercept.

Functional Properties

In osteoclast inhibition assays, adalimumab and certolizumab pegol were more effective at lower concentrations (9.6 and 14.4 nM, respectively) compared to etanercept (>130 nM). Cross-linking adalimumab enhanced its inhibitory effect two-fold, whereas etanercept required cross-linking to achieve comparable efficacy to uncross-linked adalimumab. This difference is attributable to adalimumab’s ability to form complexes with TNF, a unique mechanistic feature not shared by the receptor fusion protein etanercept.

Structural Basis for Clinical Differences

The fully humanized structure of adalimumab distinguishes it from the chimeric infliximab, potentially reducing immunogenicity. Both adalimumab and infliximab restore intracellular TNF-alpha levels to those of healthy donors through similar mechanisms, though they differ in administration routes (subcutaneous versus intravenous). The direct occupation of the receptor binding site by adalimumab, rather than partial occupation as seen with infliximab, provides a molecular basis for adalimumab’s broader clinical applications and effectiveness across multiple indications.

Clinical Implications of Mechanistic Findings

Therapeutic Efficacy Mechanisms

The multi-faceted mechanisms of adalimumab contribute to its therapeutic efficacy through complementary pathways. Steric blocking of TNF-alpha prevents receptor interaction, while cytotoxic effects eliminate pathogenic cell populations. The restoration of intracellular TNF-alpha levels to normal ranges in rheumatoid arthritis patients suggests adalimumab corrects dysregulated TNF-alpha homeostasis rather than simply blocking extracellular TNF-alpha.

In psoriasis, adalimumab’s systems-level reprogramming of keratinocyte signaling toward inflammation resolution explains its sustained clinical efficacy. The coordinated downregulation of pro-inflammatory cytokine networks and modulation of the miRNA landscape to enhance anti-inflammatory and pro-apoptotic functions provides mechanistic support for long-term treatment benefits.

Biomarker Potential

Several mechanistic findings suggest potential biomarkers for treatment monitoring. NF-κB-associated miRNAs, particularly miR-146a-5p and miR-126-3p, emerge as candidates for monitoring therapeutic response. In rheumatoid arthritis, intracellular TNF-alpha levels in CD14+ cells could serve as a biomarker, with levels correlating to EULAR-defined clinical responses. The increase in CD36 expression represents another potential marker of adalimumab activity in monocytes.

Resistance Mechanisms

The presence of anti-adalimumab antibodies represents a critical resistance mechanism. In one rheumatoid arthritis patient with neutralizing antibodies, adalimumab failed to block membrane and soluble TNF-alpha, and intracellular TNF-alpha levels did not increase. This finding highlights the importance of monitoring for neutralizing antibodies in non-responders.

Cardiovascular and Bone Protection

Mechanistic studies reveal potential benefits beyond inflammation control. In atherosclerosis models, adalimumab prevented endothelial activation, monocyte adhesion, and endothelial leakage, with abundant deposition detected in atherosclerotic plaques of hypercholesterolemic mice. The increase in CD36 expression may contribute to reduced cardiovascular risk by modulating lipid uptake and improving endothelial function, though this requires clinical validation.

The ability of adalimumab to form complexes with TNF provides sustained potency in preventing bone erosion due to chronic TNF exposure. This mechanism, mediated through restricting TNF access to TNF-RI on osteoclast precursors, explains adalimumab’s effectiveness in preventing structural damage in rheumatoid arthritis.

Anti-Aging and Cancer-Related Effects

Adalimumab’s modulation of the senescence-associated secretory phenotype (SASP) through TNF-alpha blockade reduced IL-6 release and increased eNOS expression in endothelial cells. The conditioned medium from adalimumab-treated senescent endothelial cells showed reduced capacity to promote breast cancer cell migration and mammosphere formation, suggesting potential tumor microenvironment modulation. However, significant side effects associated with TNF-alpha inhibition limit its application as an anti-aging treatment, emphasizing the need for patient selection based on risk-benefit considerations.

Synthesis

The mechanistic studies reveal adalimumab functions through five complementary mechanisms: (1) direct TNF-alpha sequestration via high-affinity binding to soluble and transmembrane forms, (2) cytotoxic elimination of transmembrane TNF-alpha-expressing cells through CDC, ADCC, and caspase-mediated apoptosis, (3) reverse signaling through transmembrane TNF-alpha inducing cell cycle arrest, (4) modulation of intracellular signaling cascades including NF-κB and MAPK pathways, and (5) epigenetic regulation through microRNA modulation.

These mechanisms explain adalimumab’s clinical advantages over other anti-TNF agents. The larger epitope interface compared to infliximab and capacity for complex formation unlike etanercept provide both higher affinity and more complete receptor blockade. The dual capacity for TNF-alpha neutralization and cytotoxic effects distinguishes adalimumab and infliximab from etanercept, explaining their superior efficacy in Crohn’s disease and Wegener’s granulomatosis.

Mechanistic heterogeneity across cell types reflects TNF-alpha’s pleiotropic roles in different tissues. In keratinocytes, adalimumab primarily modulates NF-κB signaling and microRNA networks. In monocytes, it regulates intracellular TNF-alpha production and increases CD36 expression through redox signaling. In endothelial cells, it prevents activation marker expression and reduces senescence phenotypes. In osteoclast precursors, it blocks TNF-RI access. This cell-type specificity suggests adalimumab’s therapeutic effects result from simultaneous intervention across multiple pathogenic cell populations rather than a single dominant mechanism.

The time-dependent effects also vary by mechanism and cell type. Structural binding is immediate, while effects on gene expression manifest within 2-8 hours, protein changes occur by 3-6 hours, and phenotypic changes like CD36 upregulation require 24 hours. Clinical restoration of intracellular TNF-alpha levels develops progressively over 12 injections (approximately 6 months). This temporal hierarchy indicates acute anti-inflammatory effects occur through direct TNF-alpha neutralization, while sustained therapeutic benefits depend on cellular reprogramming and restoration of homeostatic TNF-alpha regulation.

References

Shi Hu, Shuaiyi Liang, Huaizu Guo, Da-peng Zhang, Hui Li, and 7 more\ (2013).Comparison of the Inhibition Mechanisms of Adalimumab and Infliximab in Treating Tumor Necrosis Factor α-Associated Diseases from a Molecular View*. Journal of Biological Chemistry

Carlos Zamora-Atenza, C. Díaz-Torné, C. Geli, C. Díaz-López, M. A. Ortiz, and 8 more\ (2014).Adalimumab regulates intracellular TNFα production in patients with rheumatoid arthritis. Arthritis Research & Therapy

Raghav Oberoi, Jutta Schuett, H. Schuett, Ann-Kathrin Koch, M. Luchtefeld, and 2 more\ (2016).Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion. PLoS ONE

H. Mitoma, T. Horiuchi, H. Tsukamoto, Y. Tamimoto, Y. Kimoto, and 5 more\ (2008).Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor alpha-expressing cells: comparison among infliximab, etanercept, and adalimumab. Arthritis & Rheumatism

J. Boyer, Patricia Balard, H. Authier, Bruno Faucon, J. Bernad, and 5 more\ (2007).Tumor necrosis factor alpha and adalimumab differentially regulate CD36 expression in human monocytes. Arthritis Research & Therapy

Shi Hu, Shuaiyi Liang, Huaizu Guo, Da-peng Zhang, Hui Li, and 7 more\ (2014).Comparison of the inhibition mechanisms of Adalimumab and Infliximab in treating tumor necrosis factor α-associated diseases from a molecular view. Journal of Biological Chemistry

Chong Shen, G. Van Assche, P. Rutgeerts, J. Ceuppens\ (2006).Caspase activation and apoptosis induction by adalimumab: Demonstration in vitro and in vivo in a chimeric mouse model. Inflammatory Bowel Diseases

B. Harvey, Z. Kaymakcalan\ (2014).SAT0550 Inhibition of Tnf-Enhanced Human Osteoclast Function by Adalimumab is Influenced by Complex Formation in the Absence of FC Receptor Binding. Annals of the Rheumatic Diseases

F. Prattichizzo, A. Giuliani, R. Recchioni, M. Bonafè, F. Marcheselli, and 11 more\ (2016).Anti-TNF-α treatment modulates SASP and SASP-related microRNAs in endothelial cells and in circulating angiogenic cells. OncoTarget

Aleksandra Plata-Babula, Wojciech Kulej, Paweł Ordon, Julia Gajdeczka, Martyna Stefaniak, and 4 more\ (2025).Modulation of Nuclear Factor Kappa B Signaling and microRNA Profiles by Adalimumab in LPS-Stimulated Keratinocytes. International Journal of Molecular Sciences

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Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion

Raghav Oberoi, Jutta Schuett, H. Schuett, Ann-Kathrin Koch, M. Luchtefeld, K. Grote, B. Schieffer

PLoS ONE·

2016·

35 citations

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Mechanism Category

Binding/neutralization, Tissue/organ effects

Experimental Model

- Cell lines or primary cells used: THP-1 macrophages (human monocytic cell line), HUVECs (human umbilical vein endothelial cells) - Animal models: Hypercholesterolemic mice (C57BL/6J Ldlr -/-) - In vitro vs in vivo approaches: Both in vitro (cell culture experiments) and in vivo (mouse model for adalimumab deposition) - Human studies: Not applicable (no direct human study or patient population involved) - Structural studies: Not mentioned - Disease context: Atherosclerosis, rheumatoid arthritis

Key Mechanistic Findings

- Binding characteristics: Adalimumab is a humanized monoclonal antibody that binds to TNF-α, blocking its interaction with the TNF receptor. - Downstream effects on cells: Prevents activation of endothelial cells, reduces monocyte adhesion, and suppresses endothelial leakage. - Effects on protein/gene expression: Suppresses mRNA and protein expression of VCAM-1, ICAM-1, and E-selectin. - Pathway modulation: Blocks TNF-α effects on endothelial cells, reducing inflammation. - Time course of effects: Significant effects within 3 to 6 hours. - Dose-response relationships: No adverse effects on endothelial cell viability with higher concentrations.

Target Cells/Tissues

- Immune cells: THP-1 macrophages - Endothelial cells: Human umbilical vein endothelial cells (HUVECs) - Atherosclerotic tissues: Atherosclerotic plaques in hypercholesterolemic mice - Effects are direct on endothelial activation and monocyte adhesion

Anti-TNF Comparisons

- Which agents were compared: adalimumab, etanercept, infliximab, golimumab, certolizumab - Similarities and differences in mechanisms: Not mentioned - Relative potency or efficacy: Not mentioned - Unique mechanistic properties of adalimumab: First complete human monoclonal antibody with the widest range of indications - Structural or functional differences explaining clinical differences: Not mentioned

Clinical Relevance

- Mechanism explains therapeutic efficacy: Adalimumab prevents inflammatory effects of TNF-α on endothelial activation, monocyte adhesion, and endothelial leakage, extending its therapeutic options to limit vascular inflammation. - Disease-specific mechanistic relevance: Adalimumab is relevant for treating atherosclerosis due to its effects on vascular inflammation, particularly in patients with rheumatoid arthritis who have an increased risk of cardiovascular disease. - Biomarker potential: Improved endothelial function and reduced C-reactive protein levels are potential biomarkers for adalimumab's efficacy in limiting vascular inflammation. - Implications for patient selection or treatment optimization: Adalimumab may be beneficial for patients with rheumatoid arthritis at risk for cardiovascular disease, suggesting its potential in preventing or treating atherosclerosis.

Objective It is well known that atherosclerotic inflammatory vascular disease is critically driven by oxidized lipids and cytokines. In this regard, tumor necrosis factor (TNF)-α is known as a crucial mediator of early pro-atherosclerotic events. Epidemiologic data suggest that blockade of TNF-α has beneficial effects on vascular outcomes in patients with rheumatoid arthritis, however, detailed mechanistic studies are still lacking. This study aims to elucidate effects of TNF-α blockade by adalimumab–which is approved for several inflammatory disorders–on endothelial activation and monocyte adhesion under pro-atherosclerotic conditions. Methods and Results Phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice. Conclusion Our results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation.

Methods and Results

Phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophorelabelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice.

Conclusion

Our results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation.

Introduction

Atherosclerosis is a chronic inflammatory disease characterized by accumulation of lipids and fibrous elements in the large arteries and is known as the major contributor to the growing burden of cardiovascular disease (CVD) [1]. Elevated levels of blood low density lipoproteins (LDL) and endothelial dysfunction are considered as pre-disposition for atherogenesis. Hyperlipidemia can lead to accumulation of LDL molecules within the intima of arteries where it undergoes modifications such as glycation or oxidation [2]. Vascular inflammation is driven by many different cytokines predominantly derived from activated macrophages.

TNF-α is such a cytokine which has been implicated to play a key role in the pathogenesis of atherosclerosis [3] and other chronic inflammatory diseases like asthma, chronic obstructive pulmonary disease, rheumatoid arthritis and inflammatory bowel disease [4][5][6][7]. The greatest advancement in good prognosis of rheumatoid arthritis over the last decade has been made due to the identification of the pivotal role of TNF-α in its pathogenesis. Of note, although many other cytokines are involved in the progression of the disease but TNF-α has been shown to play a major role and TNF-α blockers are already approved for therapy for quite a while [8]. With regard to molecular and cellular processes, rheumatoid arthritis and atherosclerosis have much in common. Inflamed synovium and atherosclerotic plaque are similar in a number of aspects. Both scenarios are characterized by the presence of large amount of inflammatory cytokines and monocytes/macrophages [9]. In this regard, TNF-α is established as a potent inducer of endothelial and epithelial cell adhesion molecule expression such as vascular cell adhesion molecule-1 (VCAM-1) [10], intercellular adhesion molecule-1 (ICAM-1) [11] and Eselectin [12]. Studies using up-to-date medical techniques such as flow mediated dilation and laser Doppler perfusion imaging has demonstrated improved endothelial dysfunction in rheumatoid arthritis patients upon TNF-α blocker therapy [13].

Adalimumab (HUMIRA, Abbott) is one of the leading therapies for the treatment of rheumatoid arthritis. It is a humanized monoclonal antibody that binds to TNF-α and blocks its interaction with the TNF receptor [14]. It neutralizes both soluble as well as transmembrane TNF-α. Adalimumab has demonstrated a good prognosis and improvement of physical function in rheumatoid arthritis [8,15]. The important role of TNF-α for atherosclerotic plaque development in experimental models is well documented, different TNF-α-deficient mice models consistently showed reduced plaque burden [16][17][18]. However, effects of pharmacological inhibition of TNF-α on fundamental pro-atherosclerotic processes are still poorly investigated. Therefore, we investigated the potential of the TNF-α blocker adalimumab on endothelial activation with subsequent monocyte adhesion and endothelial leakage under proatherosclerotic conditions.

Mice

All experiments were approved by the governmental animal ethics committee at Philipps University Marburg and performed according to the guidelines of the Federation of European Animal Science Associations. Male C57BL/6J Ldlr -/-(B6.129S7-Ldlrtm1Her/J) mice from our own breeding mice were maintained in the Central Animal Facility at Philipps-University Marburg. Mice at the age of 8 weeks were fed a high-fat, high-cholesterol diet (D12108, Research Diets, New Brunswick, NJ) for 6 weeks. At the end of the experiment, mice were analgized and euthanized with a mixture of ketamine (120 mg/kg) and xylazine (12 mg/kg) followed by blood withdrawal from the left ventricle.

Recombinant proteins, lipoproteins and antibodies

Recombinant human TNF-α and human macrophage colony-stimulating factor (M-CSF) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Human oxidized low density lipoprotein (oxLDL), Dil(1,1´-dioctadecyl-3,3,3`,3`-tetramethylindocarbo-cyanine perchlorate)-labelled oxLDL and native LDL (nLDL) was from Kalen Biomedical LLC (Montgomery Village, MD). Antibodies for Western blot against ICAM-1, E-selectin were obtained from R&D Systems (Minneapolis, MN) and VCAM-1, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Santa Cruz (Dallas, TX). An antibody for immunofluorescence against VEcadherin was obtained from Santa Cruz. CD11b and CD11c antibodies labelled with allophycocyanin (APC) and PerCP/Cy5.5 respectively for flow cytometry were obtained from BioLegend (San Diego, SA). Appropriate secondary antibodies were from Jackson ImmunoResearch (West Grove, PA) Adalimumab (HUMIRA) was obtained from Abbott GmbH (Wiesbaden, Germany). Human IgG control antibody, 4',6-diamidino-2-phenylindole (DAPI) and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (Munich, Germany).

Cells

Human monocytic cell line (THP-1) was obtained from Cell Lines Service (Eppelheim, Germany). Cell were cultured in RPMI-1640 (Roswell Park Memorial Institute) media containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose and 1.5 g/L sodium bicarbonate. In addition, media was supplemented with 10% FCS, 1% penicillin/streptomycin (Sigma-Aldrich) and 50 μM β-mercaptoethanol (Sigma-Aldrich). Cells up to passage 20 were used for the experiments. For differentiation, THP-1 monocytes were plated at a density of 0.5x10 6 cells per milliliter in complete media supplemented with 50 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) in 6 well plates (Corning, Tewksbury, MA) and incubated for 48 hours. After incubation, cells referred to as THP-1 macrophages were adherent. Cells were washed with complete media to ensure maximal removal of PMA. After washing, cells were used for experiment.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using ficoll (Biochrom GmbH, Berlin) density gradient centrifugation. 2x10 7 PBMCs were seeded in 6 well plates (Corning) and allowed to adhere on the surface of the plates for 45 minutes in media (RPMI-Glutmax supplemented with 10% FCS and 1% penicillin/streptomycin). Non-adhered cells were removed with warm PBS, media containing recombinant human M-CSF (50 ng/mL) was added and cells were cultured for 8 days and subsequently used for experiments. Medium was changed on day 4 and 6.

Human endothelial cells (human umbilical vein endothelial cells, HUVECs) were obtained from Promocell (Heidelberg, Germany). Cells were cultured in endothelial growth medium (EGM; Promocell) supplemented with 2% fetal calf serum (FCS, PAN Biotech, Aidenbach, Germany), growth factors (epidermal growth factor, vascular endothelial growth factor 165, basic fibroblast growth factor, insulin-like growth factor) and heparin in 75 cm 2 cell culture flasks (Sarstedt, Nürnbrecht, Germany). Cells were kept in endothelial cell basal medium (EBM, Promocell) supplemented with 1% FCS without growth factors before and during experiments. Cells between passage 2 and 4 were used for the experiments.

Tissue preparation

Preparation of mouse tissues was performed as described previously [19]. Briefly, after withdrawal of blood from the left ventricle, heart and aorta were removed after perfusion with PBS. The thoraco-abdominal aorta was fixed in 3.7% formalin for the measurement of atherosclerotic burden. Liver and spleen were embedded and snap-frozen in Tissue Tek OCT (Sakura Finetek, Staufen, Germany) for immunohistochemistry.

Fluorescence labeling of adalimumab

In order to analyze the vascular distribution of adalimumab, we labelled the substance using the DyLight™ 549 Microscale Antibody Labeling Kit according to the manufacturer's protocol (Thermo Scientific, Rockford, IL). Briefly, one vial of DyLight™ 549 Reagent was dissolved in 1 mg adalimumab solution (in PBS; PAA Laboratories, Colbe, Germany) and purified with a resin spin column to remove excess fluor. Hypercholesterolemic Ldlr -/-mice were injected with fluorophore-labelled adalimumab (8.0 mg/kg in 200 μl PBS i.p.) for two consecutive days and sacrificed 12 hours after last injection. Vehicle injection (200 μl PBS) was used as control. Subsequently, the thoraco-abdominal aorta was removed, opened longitudinally, pinned on a black silicone-covered dish under a stereomicroscope (Stemi DV4, Carl Zeiss Microimaging, Jena, Germany). Subsequently, it was photographed under an epi-fluorescence microscope using appropriate filter sets from Axio Vert.A1 microscope with an AxioCam MRm camera (Carl Zeiss, Jena, Germany).

Histochemistry and immunohistochemistry

Atherosclerotic burden of the thoraco-abdominal aorta were visualized by en face oil red Ostaining as described previously [19]. After epi-fluorescence microscopy the pinned thoracoabdominal aorta was formalin-fixed and stained with propylene glycol-dissolved oil red O (2 hours at room temperature). The pinned aorta was photographed under PBS immersion using a stand-equipped camera (EOS 600D, Canon, Tokyo, Japan).

Serial cryostat sections of liver and spleen tissue (8 μm, HM-500 O cryostat, Microm, Walldorf, Germany) were produced and stored at -20°C. For staining, sections were air-dried, fixed in ice-cold acetone and stained with an anti-human IgG Fc antibody (Rockland, Gilbertsville, PA) or corresponding IgG control (Santa Cruz) and with a horseradish peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany), followed by incubation with DAB substrate (Vector Laboratories, Burlingame, CA) and counter stained with hematoxylin (Sigma-Aldrich).

Flow cytometry

Differentiation of THP-1 macrophages was analyzed using flow cytometry. Briefly, THP-1 monocytes differentiated with PMA or vehicle control dimethyl sulfoxide (DMSO, Sigma-Aldrich) were stained for 20 min on ice for the THP-1 macrophage maturation marker CD11b and CD11c antibodies labelled with APC and PerCP/Cy5.5, respectively. Appropriate IgG-APC was used as isotype control. Cells were acquired on FACS LSR II flow cytometer (BD Biosciences, San Jose, CA) and analyzed using flowjo (Tree Star Inc., Ashland, OR).

Preparation of conditioned media

THP-1 macrophages were stimulated with 25 and 50 μg/mL oxLDL for 48 hours. After stimulation, supernatant was collected, pre-cleared by centrifugation and passed through 0.2 μm filters and kept at 4°C. This supernatant is referred to as oxLDL conditioned media (oxLDL CM), conditioned media from unstimulated THP-1 macrophages were used as control (control CM).

Real-time polymerase chain reaction (PCR)

0.5x10 5 HUVECs were plated in 12-well plates (Corning) in 1 ml EGM per well and grown till confluency. Cells were starved in EBM with 1% FCS for 6 hours. After starvation, cells were stimulated with conditioned media with or without adalimumab or IgG (both 1 μg/mL) for the mentioned time. Total RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany) and reverse-transcribed with SuperScript reverse transcriptase kit (Applied Biosystems, Darmstadt, Germany). Real Time PCR was performed in duplicates in a total volume of 20 μL using Power SYBR1 green PCR master mixture (Applied Biosystems) on a Step OnePlus Real-Time PCR system (Applied Biosystems) in 96-well PCR plates (Applied Biosystems). Real-time PCR was done with an initial denaturation step at 95°C for 10 min followed by 40 PCR cycles consisting of 95°C for 15 s, 60°C for 1 min, and 72°C for 1 min, and SYBR green fluorescence emission were monitored after each cycle. For normalization, expression of β-actin was determined in duplicates. Fold change with respect to control was calculated using the 2 -ΔΔCT method. PCR primers were obtained from TIB MOLBIOL (Berlin, Germany). Primer sequences are as follows: VCAM-1 forward: 5'-TGT TTG CAG CTT CTC AAG CTT TT-3', VCAM-1 reverse: 5'-GAT GTG GTC CCC TCA TT CGT-3', ICAM-1 forward: 5'-AGC TTC GTG TCC TGT ATG GC-3', ICAM-1 reverse: 5'-TTT CTG GCC ACG TCC AGT TT-3', E-selectin forward: 5'-GCC TGC AAT GTG GTT GAG TG-3', E-selectin reverse: 5'-ACG AAC CCA TTG GCT GGA TT-3', P-selectin forward: 5'-TGA TAA TGG GTG GGA CGC TC-3', P-selectin reverse: 5'-TTT ATG GAA ACC TTA AGG ACT CGG G-3', CD36 forward: 5'-GGC CAT ACA CCT ACA GGG AAC-3', CD36 reverse: 5'-GCA CCT GGG ACC ACT CTA TG-3', LOX1 forward: 5'-CAG CCC CAT CCA GAA TGG AAA-3', LOX1 reverse: 5'-TCG GGC TCA TTT AAC TGG GAA-3', β-actin forward: 5'-CAT GTA CGT TGC TAT CCA GGC-3', β-actin reverse: 5'-CTC CTT AAT GTC ACG CAC GAT-3'.

Western blotting 5x10 5 HUVECs were plated in 60 mm dishes (Corning) in 4 ml EGM per well and grown till confluency. Cells were starved as mentioned before. After starvation, cells were stimulated with conditioned media with or without adalimumab (1 μg/mL) for 6 hours. IgG (1 μg/mL) was used as isotype control. Total protein was extracted with buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris supplemented with protease inhibitor cocktail (Roche, Penzberg, Germany). Total protein content was measured using the BCA kit (Thermo Scientific, Waltham, MA) according to manufactures protocol. 30 μg of total protein was loaded onto 10% denaturing SDS gel and transferred to 0.45 μm polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Amersham, UK) for immunoblotting. Membrane was blocked with 5% non-fat dry milk, (Sigma-Aldrich) and probed with primary antibody against proteins of interest followed by horseradish peroxidase (HRP)-labelled secondary antibody. Proteins were detected using chemiluminescence substrate (BioRad Laboratories, Hercules, USA). Results were documented on a Chemo-star imaging system (INTAS, Göttingen, Germany). Signal intensity of chemiluminescence was quantified using Quantity One software (BioRad).

Enzyme-linked immunosorbent assay (ELISA)

Supernatants from THP-1 macrophages and primary human macrophages after oxLDL stimulation were analyzed for TNF-α by ELISA from R&D Systems according to the manufacturers' protocols and analyzed on an Infinite M200 pro plate reader (TECAN Instruments, Maennedorf, Switzerland). LPS (100 ng/mL) was used as positive control.

Protein array

Supernatants from THP-1 macrophages after stimulation with oxLDL and with or without adalimumab (1 μg/mL) were analyzed with commercially available protein array for human cytokines and growth factor related to inflammation according to the manufacturer's instructions (RayBiotech, Norcross, GA). Briefly, antibody array membranes were blocked for 30 min with blocking buffer followed by incubation with 1 mL of undiluted supernatants overnight at 4°C. After washing, membranes were incubated with detection antibody cocktail overnight at 4°C, followed by incubation with HRP-streptavidin for again overnight at 4°C. Membranes were developed using chemiluminescence detection method and results were documented on a Chemo-star imaging system (INTAS). Signal intensity of chemiluminescence was quantified using Quantity One software (BioRad Laboratories).

Cell cytotoxicity (alamar blue) 1 x 10 4 HUVECs were plated per well of 96 well plate (Corning) in EGM. After reaching subconfluency cells were cultured in different concentration of adalimumab (0.01-10 μg/mL), IgG isotype control (10 μg/mL) and 0.1% Triton-X (Sigma-Aldrich) as positive control in 5% CO 2 at 37°C. After 12 hours, 10% Alamar blue (Pierce, Thermo Scientific) was added to each well and kept in incubator for another 6 hours. Absorbance of oxidized alamar blue was measured at 600 nm on Infinite M200 pro plate reader (TECAN Instruments). All measurements were performed in triplicate.

Foam cell formation

Foam cell formation of THP-1 macrophages was analyzed by their ability to take up oxLDL labelled with the lipophilic tracer Dil. Therefore, PMA-differentiated THP-1 macrophages were incubated with Dil-labelled oxLDL or Dil-labelled nLDL (each 10 μg/mL) for 4 hours in RPMI-1640 supplemented with 1% FCS. Nuclei were visualized using DAPI. Foam cell formation was evaluated by FACS (LSR II flow cytometer) and by a DM550B fluorescence microscope with a DFC300FX camera (Leica Microsystems, Wetzlar, Germany).

Adhesion assay

Adhesion under static conditions. 3x10 4 HUVECs were plated per well of a 48 well plate (Corning) in EGM and grown to complete confluence. Cells were starved in EBM supplemented with 1% FCS for 6 hours. Following starvation, cells were incubated with conditioned media with or without adalimumab (1 μg/mL) for 4 hours. TNF-α (10 ng/mL) was used as positive control and IgG (1 μg/mL) was used as isotype control. In parallel THP-1 monocytes were labelled with 5 μM of CellTracker™ green (Invitrogen) according to the manufacturer's instructions. After stimulation, HUVECs were washed twice with 500 μL EBM and 0.5x10 6 labelled THP-1 monocytes were added per well of a 48 well plate and incubated for 30 min in 5% CO 2 at 37°C. After incubation each well was washed three times with 500 μL EBM and 10 high power field (HPF) digital images were taken using Axio Vert.A1 microscope with an AxioCam MRm camera (Carl Zeiss, Jena, Germany). Adhered cells per HPF was counted using ImageJ software (National Institute of Health, USA).

Adhesion under flow conditions. Flow based adhesion assay was done as described by Shetty et al. [20]. Briefly, 5x10 5 HUVECs were plated in μ-Slide I 0.4 flow chambers (ibidi GmbH, Munich, Germany) in EGM and grown to complete confluence. Cells were starved in EBM supplemented with 1% FCS for 6 hours. Following starvation, cells were incubated with conditioned media with or without adalimumab (1 μg/mL) for 4 hours. TNF-α (10 ng/mL) was used as positive control and IgG (1 μg/mL) was used as isotype control. After stimulation, μ-Slide was connected to a 20 cm silicon tubing attached to a luer lock adaptor (B Braun AG, Melsungen, Germany) which was in turn connected to a 50 mL syringe attached to a Perfusor VII pump (B Braun AG). For flow conditions, a flow rate of 0.53 mL/min (which corresponds to laminar flow of 0.5 dyne/cm 2 ) was maintained. Endothelial layer was perfused with EBM for 2 min to remove any debris and dead cells. After washing, THP-1 monocytes at a concentration of 1x10 6 cells/mL were perfused for 5 min at a constant shear stress of 0.5 dyne/cm 2 , followed by perfusion with EBM for 5 min to remove unbound cells. The last 2 min of washing with EBM were recorded by a DM550B fluorescence microscope with a DFC300FX camera (Leica Microsystems) and 20 HPF digital images from this recording were subsequently used for the analysis. Adhered cells per high power field were counted using ImageJ software.

Migration assay

Transwell inserts (Corning) with 8 μm pores were coated with fibronectin (20 μg/mL, Roche, Germany) for 30 min at 37°C. 700 μL of conditioned media was added to each well of a 24 well plate (Corning) followed by addition of fibronectin coated inserts. To each insert 1x10 5 THP-1 monocytes were added and cells were allowed to migrate for 12 hours in 5% CO 2 at 37°C. TNF-α (10 μg/mL) was used as control and IgG (1 μg/mL) was used as isotype control. For staining inserts were washed with phosphate buffered saline (Sigma-Aldrich) followed by fixation with 3.7% formalin solution (Roth) for 10 min. Following fixation inserts were stained with 0.2% crystal violet (Sigma-Aldrich). Cells on the top of the insert were removed using a cotton swab and only cells which migrated across the membrane were analyzed. The membrane of the transwell insert were carefully cut using a scalpel and mounted on a glass slide using mounting medium (VectaMount™ AQ, Vector Laboratories). 10 high power field (HPF) digital images were taken using DMI 3000B microscope with a DFC300FX camera (Leica Microsystems). Cells per HPF were counted using ImageJ software.

Permeability assay

Permeability of HUVECs cultured on transwell inserts was assayed using Evans blue bound to bovine serum albumin (BSA). Briefly, 2x10 4 HUVECs were cultured on transwell inserts (Greiner bio-one, Frickenhausen, Germany) with 0.4 μm pore size in EGM. At day 3, EGM in the luminal (upper) chamber was replaced by conditioned media with or without adalimumab or IgG (1 μg/mL) for 4 hours. Following stimulation, inserts were washed with PBS with Ca ++ and Mg ++ . Afterwards, 300 μL of 0.002% Evans blue bound to 0.1% BSA (Sigma-Aldrich) in PBS with Ca ++ and Mg ++ was added to the luminal chamber and 1 mL PBS with Ca ++ and Mg ++ was added to the abluminal (lower) chamber. After 3 hours, 100 μL from the abluminal chamber was removed and absorbance was measured at 620 nm using Infinite M200 pro plate reader (TECAN Instruments). TNF-α (10 ng/mL) was used as control.

In parallel, 5x10 4 HUVECs were cultured on chamber slides (Sarstedt) and treated as mentioned before. Afterwards, cells were fixed in 4% paraformaldehyde for 10 min, blocked with 1% BSA, and stained with an antibody against VE-cadherin at +4°C overnight followed by an appropriated TRITC-labelled secondary antibody. Nuclei were visualized using DAPI. Coverslips were mounted onto glass slides using mounting medium (VectaShield 1 , Vector Laboratories). Images were captured using a DM550B fluorescence microscope with a DFC300FX camera (Leica Microsystems).

Statistical analysis

All data are represented as mean with standard error of the mean (SEM). Data were compared using the 2-tailed Student t test for independent samples or one-way ANOVA followed by Tukey multiple comparison test when more than two groups were compared (GraphPad Prism, version 6.05, GraphPad Software, Inc., USA. P value of less than 0.05 was considered statistically significant. Numbers of replicated experiments are indicated in each figure legend. RT-PCR and ELISA measurements were performed in duplicates.

OxLDL-dependent release of TNF-α and other inflammatory factors from THP-1 macrophages

Deposition of oxLDL in the arterial wall and subsequent vascular invasion of monocytes which differentiate into macrophages and release cytokines such as TNF-α is known as a key event in atherosclerotic plaque development [1,2,21]. To mimic these processes, we differentiated human monocytic THP-1 cells by treatment with PMA for 48 hours into macrophages (S1A Fig) . Cells became adhered and up-regulated the THP-1 macrophage maturation markers CD11b and CD11c as confirmed by flow cytometry (S1B Fig) [22] as well as the scavenger receptors CD36 and LOX1 as determined by RT-PCR (S1C Fig) As already reported by others [23], analysis of the conditioned medium (hereinafter referred to as oxLDL CM and control CM) by ELISA revealed an oxLDL-induced TNF-α release from THP-1 macrophages (Fig 1A ) that was confirmed in additional control experiments using primary human macrophages (Fig 1B ). To explore which other pro-inflammatory factors were oxLDL-dependently released from THP-1 macrophages we took advantage of protein arrays. Additional prominent cytokines and chemokines were identified which were at least 2-fold induced, e.g. GROα, IL-6, MCP-1 (Fig 1C ).

The TNF-α inhibitor adalimumab suppresses endothelial activation by oxLDL-stimulated THP-1 macrophages

Using the before generated oxLDL CM we studied early pro-atherosclerotic processes and a potential intervention with the TNF-α inhibitor adalimumab on these processes. First, we investigated endothelial activation which is such an early event. As expected, oxLDL CM was found to be highly potent to facilitate endothelial activation as shown by enhanced mRNA expression of major adhesion molecules in HUVECs. The mRNA expression of VCAM-1, ICAM-1 and E-selectin was significantly up-regulated 3 to 6 hours after stimulation-a process known to be stimulated by TNF-α [10][11][12]-whereas mRNA expression of P-selectin was unchanged (Fig 2A -2D ). Accordingly, inhibition of TNF-α by addition of adalimumab to the oxLDL CM significantly prevented the up-regulated mRNA expression as well as protein expression of the endothelial adhesion molecules VCAM-1, ICAM-1 and E-selectin in HUVECs after 6 hours (Fig 3A -3G ). Of note, treatment of HUVECs with adalimumab for up to 12 hours shows no adverse effect on their cell viability, even with a 10-fold higher concentration of adalimumab than used in this study (S2 Fig).

The TNF-α inhibitor adalimumab suppresses adhesion of THP-1 monocytes to endothelial cells

Since TNF-α-induced expression of adhesion molecules is critically involved in the attachment of monocytes to the endothelium during the initial phase of atherosclerotic plaque development [10,24] we next investigated the adhesion of THP-1 monocytes to a confluent monolayer of HUVECs under static conditions as well as under in vivo even more relevant flow conditions. Adhesion of THP-1 monocytes was strongly enhanced when HUVECs were incubated with oxLDL CM which was completely blocked by adalimumab (Fig 4A , 4B, 4D and 4E ). As expected, recombinant TNF-α significantly enhanced THP-1 monocyte adhesion (Fig 4C and 4F ). Next we investigated migration of THP-1 monocytes using the transwell system. We observed a significant increase in THP-1 monocyte migration in response to oxLDL CM but no inhibitory effect with adalimumab (Fig 5A and 5B ). Accordingly, we did not observe migration of THP-1 monocytes using TNF-α as a control (Fig 5C ) indicating that maybe other ox-LDL-induced factors from THP-1 macrophages (Fig 1B ) were responsible for this effect. The TNF-α inhibitor adalimumab inhibits endothelial leakage Besides adhesion molecule expression, increased endothelial permeability with increased leucocyte invasion and augmented lipid uptake into the diseased vessel wall is another well-known process in early atherosclerosis and likewise promoted by TNF-α [25,26]. Using the oxLDL CM, we next investigated its effect on endothelial permeability again taking advantage of the transwell system. As observed, oxLDL CM enhanced the vascular permeability of a confluent endothelial monolayer which resulted in increased diffusion of Evan's blue bound BSA from the luminal to the abluminal chamber. Inhibition of TNF-α by adalimumab significantly inhibited Evan´s blue perfusion (Fig 6A ). Consequently, we observed enhanced endothelial permeability with TNF-α (Fig 6B ). In addition, we visualized endothelial cell-to-cell adherens junctions by vascular endothelial (VE)-cadherin staining. Immunofluorescence shows loss of endothelial cell integrity with partial gap formation of the monolayer in response to oxLDL CM which was prevented by adalimumab (Fig 6C ).

Discussion

We here report that the TNF-α antagonist adalimumab effectively blocks effects of TNF-α released from oxLDL-stimulated THP-1 macrophages on endothelial cells, i.e. increased endothelial adhesion molecule expression, subsequent monocyte adhesion and endothelial leakage (S4 Fig) . These processes are known as early events in atherosclerotic plaque development which potentially renders adalimumab eligible for the treatment of vascular inflammation.

Hyperlipidemia is known as a crucial trigger for atherosclerotic vascular disease. Augmented cholesterol deposition in the vascular wall and particularly subsequent oxLDL accumulation is an early key event of atherosclerotic plaque formation [1,2]. In this regard, oxLDL promotes directly the recruitment and retention of monocytes into the vessel wall which consecutively differentiate into tissue macrophages. However, oxLDL also induces the secretion of high amounts of pro-inflammatory cytokines and chemokines from these cells which further amplifies vascular inflammation and plaque progression [27]. TNF-α is one of such cytokines which is released from macrophages upon oxLDL incubation [23] and fires many pro-inflammatory processes in early atherosclerosis [3]. The crucial role of TNF-α in atherosclerosis is well supported by scientific evidence. Disruption of the TNF-α gene locus in different experimental mouse models consistently revealed diminished lesion progression [16][17][18] and demonstrated that TNF-α is actively involved in the progression of atherosclerosis. However, little is known if pharmacological TNF-α inhibitors could be successfully used to limit progression of atherosclerosis in experimental models. To our best knowledge, so far just two studies exist using such an approach. Brånén et al. treated Apoe -/-mice with recombinant soluble TNF receptor I and observed 75% reduction in lesion size [16]. In addition, Tuleta and co-workers found improved endothelial function and reduced atherosclerotic plaques in Apoe -/-mice subjected to chronic intermitted hypoxia and treated with the TNF-α inhibitor infliximab [28].

In this regard, TNF-α has been found to play not only a pivotal role in the pathogenesis of atherosclerosis [3] but also in many other chronic inflammatory diseases, e.g. asthma, chronic obstructive pulmonary disease, rheumatoid arthritis and inflammatory bowel disease [4][5][6][7]. Thus, it is not surprising that TNF-α agonist have already entered clinic routine a while ago. These pharmacological agents include etanercept, a soluble TNF-α receptor antagonist and antibodies targeting TNF-α, i.e. adalimumab and golimumab a fully human monoclonal antibody, infliximab, a chimeric monoclonal antibody and certolizumab a humanized F ab fragment linked to polyethylene glycol [8,29]. These drugs have in common that they efficiently block In the present study we used adalimumab which was the first complete human monoclonal antibody and has the widest range of indications among all TNF-α inhibitors. Adalimumab is approved for the treatment of inflammatory THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) for 4 hours followed by the addition of CellTracker green-labelled THP-1 monocytes. Pictures before and after washing with basal medium are shown. (B) Adherent cells per high power field were quantified after washing. (C) HUVECs monolayer was stimulated with TNF-α (10 ng/mL) for 4 hours followed by the addition of CellTracker green-labelled THP-1 monocytes. Adherent cells per high power field were quantified after washing. Adhesion assay under flow conditions. (D) Phase contrast images of THP-1 monocytes on a HUVECs monolayer after incubation with conditioned media from oxLDL-stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) for 4 hours followed by the addition of THP-1 monocytes with a flow rate of 0.53 mL/min (0.5 dyne/cm 2 ). (E) Adherent cells per high power field were quantified after washing. (F) HUVECs monolayer was stimulated with TNF-α (10 ng/mL) for 4 hours followed by the addition of THP-1 monocytes with a flow rate of 0.53 mL/ min (0.5 dyne/cm 2 ). Adherent cells per high power field were quantified after washing. Conditioned medium from unstimulated THP-1 macrophages (control CM), IgG isotype (1 μg/mL) and medium were used as diseases such as rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, inflammatory bowel diseases (Crohn's disease, ulcerative colitis, pediatric Crohn's disease, and intestinal Behçet's disease), ankylosing spondylitis, axial spondyloarthritis and juvenile idiopathic arthritis [30].

Of all these inflammatory diseases rheumatoid arthritis share the highest similarity to atherosclerosis [9], but a large-scale clinical trial addressing a potential benefit of adalimumab for patients with coronary artery disease or peripheral artery disease has not yet been carried out. In other words, little is known so far about the effect of adalimumab on atherosclerosis. Of note, patients with rheumatoid arthritis have an increased prevalence of atherosclerosis [31]. However, with rheumatoid arthritis patients on adalimumab therapy, there are several smaller studies existing showing promising results on study endpoints related to early atherosclerotic vascular disease. For example, adalimumab therapy is associated with improvement of endothelial function, arrest of intima-media wall thickening and decreased C-reactive protein levels [32][33][34]. To date, the underlying mechanism of TNF-α inhibition by adalimumab on sub-clinic atherosclerosis is not fully comprehended. In this regard, a number of molecular and cellular processes of early atherosclerotic events are known to be crucially promoted by TNF-α. These include up-regulation of adhesion molecules [10][11][12] subsequent monocyte adhesion [10,24] and increased endothelial permeability resulting in disruption of endothelial barrier function doi:10.1371/journal.pone.0160145.g006 [25,26]. In our present study, we used human cell culture models and show that all these processes could by suppressed by adalimumab. Finally, we took advantage of an experimental murine model of early atherosclerosis and observed enriched fluorescent-labelled adalimumab mainly in atherosclerotic plaques which documents good penetration of the drug into the tissue of our interest. Our results demonstrate that adalimumab is capable of blocking TNF-α -which is released in high amounts from plaque macrophages in response to oxLDL-and its downstream pro-atherosclerotic effects on endothelial cells and monocytes.

Conclusion

Since adalimumab is an exclusive human specific TNF-α inhibitor we could not test its effect on atherosclerotic plaque development in an experimental mouse model of atherosclerosis. In our in vitro studies, we have identified effects of adalimumab to prevent fundamental inflammatory processes such as up-regulation of endothelial adhesion molecules, subsequent monocyte adhesion and endothelial leakage, suggesting a potential inhibitory influence of adalimumab on atherosclerotic plaque development. Accordingly, adalimumab-which is widely used for the therapy of patients with rheumatoid arthritis-likewise limits vascular inflammation which is of particular interest because these patients have an increased risk for cardiovascular disease.

availability

All relevant data are within the paper and its Supporting Information files.

Acknowledgements

AcknowledgmentsWe are grateful to Dr. Katrin Roth (Microscopy Core Facility, Philipps-University Marburg) for her support to set up the recording of the adhesion experiments under flow conditions and we thank Daniela Beppler for her excellent technical assistance.

Funding

This study was funded by AbbVie Germany.The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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