Elicit: Etanercept: TNF Binding and Signaling Effects

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Etanercept: TNF Binding and Signaling Effects

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May 5, 2026

Review etanercept's TNF-binding mechanism and downstream signaling effects

Etanercept binds soluble TNF in a 1:1 ratio through its p75 receptor structure, activating NF-κB and inflammatory gene transcription but lacking the reverse signaling, TGF-β induction, and apoptosis-inducing capabilities of monoclonal antibody TNF antagonists.

Abstract

Etanercept is a dimeric soluble p75 TNF receptor that binds TNF in a 1:1 stoichiometric ratio, forming relatively unstable complexes with soluble TNF and uniquely binding lymphotoxin-alpha. This structural configuration differs fundamentally from monoclonal antibody TNF antagonists like adalimumab and infliximab, which bind in 2:1-3:1 ratios and form stable complexes. Etanercept exhibits approximately 13-fold higher clearance than adalimumab or infliximab, and its complexes with TNF maintain a monomeric binding profile without enhanced binding to low-affinity Fc-gamma receptors, resulting in less efficient internalization and clearance by cells compared to adalimumab:TNF complexes.

Downstream signaling effects include activation of TNFR1 and TNFR2 pathways, leading to NF-κB activation and transcriptional upregulation of inflammatory genes including IL-8, IL-1, IL-6, COX2, and TNF. However, etanercept’s unstable complex formation means it does not fully engage these pathways as effectively as monoclonal antibodies. Critically, etanercept fails to induce TGF-β production in human macrophages and does not trigger effective reverse signaling through membrane-bound TNF, unlike infliximab which induces IL-10 expression, reactive oxygen species accumulation, and apoptosis. Etanercept does not induce apoptosis in lymphocytes from inflammatory bowel disease patients and lacks antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity capacity due to its receptor-based structure. These mechanistic limitations explain etanercept’s clinical efficacy in rheumatoid arthritis, where soluble TNF predominates, but its failure in Crohn’s disease, where membrane-bound TNF and reverse signaling are pathogenically important.

Methods

We analyzed 10 sources from an initial pool of 200, using 7 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Etanercept as Primary Intervention, TNF-Related Mechanisms, Mechanistic Outcomes, Appropriate Study Design, Etanercept Inclusion, Study Type Quality, TNF Pathway Focus

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: “Review etanercept’s TNF-binding mechanism and downstream signaling effects”

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.

Extract the study design and experimental approach used to investigate etanercept’s TNF-binding mechanism and/or downstream signaling effects, including:

Extract all details about how etanercept binds to TNF, including:

Extract all signaling pathways and cellular processes affected by etanercept’s TNF binding, including:

Extract the specific cellular and biological outcomes resulting from etanercept’s mechanism of action, including:

Extract any information about etanercept’s effects on transmembrane TNF-α reverse signaling, including:

Extract how etanercept’s TNF-binding mechanism and downstream effects differ from other TNF antagonists (adalimumab, infliximab, etc.), including:

Extract the specific experimental techniques used to study etanercept’s mechanism and signaling effects, including:

Extract the main conclusions about etanercept’s TNF-binding mechanism and downstream signaling effects, including:

Results

Characteristics of Included Studies

Study

Full Text Retrieved?

Study Type

Research Focus

Cell/Model System

D. Furst et al., 2006

No

Literature review

Mechanisms of TNF antagonists and granulomatous infection risk

Not applicable

B. Harvey & Z. Kaymakcalan, 2014

No

In vitro, mechanistic

Osteoclast function inhibition by TNF antagonists

Primary human osteoclast precursors

B. Harvey et al., 2018

Yes

In vitro

FcγR-mediated clearance of TNF:biologic complexes

CHO cells with human FcγRs, primary human osteoclast precursors

B. Harvey et al., 2020

No

In vitro proteomics

Proteomic profiling of TNF-activated osteoclasts

Human bone marrow-derived osteoclast precursors (3 donors)

B. Harvey et al., 2016

No

In vitro proteomics

Proteomic changes in TNF-stimulated osteoclasts

Human osteoclast precursors

K. Papp et al., 2007

No

Review

Mechanism, pharmacokinetics, and drug interactions

Not applicable

U. Billmeier et al., 2016

Yes

In vitro, mechanistic

Molecular mechanisms in inflammatory bowel disease

PBMCs, cell lines expressing mTNF

Z. Szondy & Anna Pallai, 2017

No

Review

Transmembrane TNF-α reverse signaling

Not applicable

A. Tuttolomondo et al., 2014

No

Review of animal models

TNF inhibition in brain injury

Animal models of ischemic and traumatic neuronal damage

Heejin Lim et al., 2018

No

Review

Structural biology of TNFα antagonists

Not applicable

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Pageof

The included studies comprised four reviews, one review of animal models, and five in vitro mechanistic studies. Only two papers had full text available (B. Harvey et al., 2018 and U. Billmeier et al., 2016). The in vitro studies predominantly used human osteoclast precursors as experimental models, with experimental durations ranging from 5-6 days. Sample sizes in the proteomics studies were small, with three donors in the 2020 study.

Thematic Analysis

TNF-Binding Mechanism

Etanercept is a dimeric soluble form of the p75 TNF receptor that binds TNF in a 1:1 stoichiometric ratio. This contrasts fundamentally with monoclonal antibody TNF antagonists like infliximab and adalimumab, which bind in 2:1 to 3:1 ratios. Etanercept forms relatively unstable complexes with soluble TNF, whereas infliximab forms stable complexes. Despite this instability, etanercept exhibits similar affinity to golimumab and greater avidity for soluble TNF compared to adalimumab and infliximab.

A unique property of etanercept is its ability to bind lymphotoxin-alpha, which may enhance its efficiency at preventing granuloma formation compared to infliximab. When etanercept forms complexes with TNF, these complexes maintain a monomeric binding profile with no gain of binding to low affinity Fc-gamma receptors, unlike adalimumab:TNF complexes which acquire enhanced binding to these receptors.

The clearance of etanercept is approximately 13 times higher than infliximab or adalimumab. This rapid clearance, combined with subcutaneous administration (versus intravenous for infliximab), results in lower peak concentrations, potentially contributing to differences in therapeutic effects and safety profiles.

Downstream Signaling Pathways

Activation of TNF receptors by etanercept-bound TNF initiates intracellular signaling cascades involving apoptosis, cell proliferation, and cytokine secretion. The TNFR1 pathway activates NF-κB, leading to transcriptional upregulation of genes including IL8, IL1, IL6, COX2, and TNF. TNFR1 can also activate a Caspase8-dependent pathway resulting in apoptosis. The TNFR2 pathway is involved in proliferation, migration, and cytokine production, and can mediate reverse signaling.

However, etanercept’s formation of unstable complexes with soluble TNF means it does not fully engage signaling pathways as effectively as other anti-TNF agents. Notably, etanercept fails to induce TGF-β in human macrophages, representing a significant mechanistic difference from infliximab and adalimumab. This inability to induce TGF-β production is linked to etanercept’s failure to trigger effective reverse signaling through membrane-bound TNF.

In the central nervous system, etanercept influences neuroinflammatory pathways through inhibition of microglia and astrocyte activation. It modifies blood-brain barrier permeability and affects glutamatergic transmission and synaptic plasticity. Etanercept decreases AMPA receptor density and increases γ-aminobutyric acid receptor expression by blocking TNF-α’s effects on these receptors.

In osteoclastogenesis, etanercept affects three major pathways: receptor-mediated endocytosis (involving CD163 and IGFBP2), oxidation-reduction processes (involving FABP4), and cell adhesion (involving ICAM-1 and TGFBI). However, etanercept is less effective than adalimumab at restoring these pathway-associated proteins to baseline levels.

Cellular Effects

Etanercept does not induce apoptosis in gastrointestinal mucosa, unlike infliximab, though both may cause apoptosis in synovium at steady state. In lymphocytes from Crohn’s disease patients, etanercept fails to induce apoptosis, which represents a key mechanistic limitation. Etanercept does not inhibit IFN-gamma expression, contrasting with adalimumab and infliximab.

In the osteoclast system, etanercept reduces TNF-enhanced osteoclast function, though less effectively than adalimumab or certolizumab pegol at lower concentrations. The mechanism involves restricting TNF access to TNF-RI. Etanercept does not reduce pro-osteoclastogenic factors like IGFBP2 and ICAM-1 as effectively as adalimumab, and it shows intermediate effects on cell adhesion pathways. Etanercept also fails to restore CD163 levels (an M2 macrophage polarization marker) as effectively as adalimumab.

In inflammatory mediator production, etanercept partially inhibits IL-1 beta release but does not affect IL-10 or IL-12 expression. In the brain, etanercept decreases microglia activation, influences blood-brain barrier permeability, and modulates activation of microglia and astrocytes.

Reverse Signaling Effects

Etanercept does not trigger reverse signaling in the same manner as infliximab and golimumab. While infliximab induces E-selectin expression, IL-10 expression, ROS accumulation, and apoptosis through reverse signaling, etanercept induces only E-selectin expression but not the other downstream effects. Critically, etanercept fails to induce TGF-β production in human macrophages, a downstream effect of reverse signaling mediated by membrane-bound TNF. This suggests etanercept does not effectively engage reverse signaling pathways associated with transmembrane TNF-α.

One review suggested that etanercept triggers reverse signaling leading to TGF-β production in macrophages, though this finding is contradicted by experimental data showing etanercept’s failure to induce TGF-β.

Comparative Mechanisms with Other TNF Antagonists

Characteristic

Etanercept

Adalimumab/Infliximab

Source

Molecular structure

Soluble TNF receptor

Monoclonal antibodies

Furst et al., 2006

Binding stoichiometry

1:1 ratio

2:1 to 3:1 ratios

Furst et al., 2006

Complex stability

Unstable complexes with sTNF

Stable complexes

Billmeier et al., 2016

Lymphotoxin-α binding

Yes

No (infliximab)

Furst et al., 2006

Antibody-mediated cell lysis

No

Yes

Furst et al., 2006

IFN-gamma inhibition

No

Yes

Furst et al., 2006

Complex formation with TNF

Does not form antibody-type complexes

Forms complexes

Harvey & Kaymakcalan, 2014

FcγR binding profile

Monomeric profile, no gain on low affinity FcγR

Enhanced binding to low affinity FcγR

Harvey et al., 2018

Internalization by osteoclasts

1.5-fold reduction in mature osteoclasts

4-fold reduction (adalimumab)

Harvey et al., 2018

TGF-β induction

No

Yes (infliximab, adalimumab)

Billmeier et al., 2016

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The fundamental structural difference between etanercept (a TNF receptor:Fc fusion protein) and monoclonal antibodies like adalimumab underlies many of the mechanistic distinctions. Etanercept cannot form the same type of complexes with TNF that adalimumab forms, which appears central to therapeutic differences in osteoclast inhibition. When adalimumab forms complexes with TNF, these complexes are cross-linked and show enhanced inhibitory effects, whereas etanercept complexes do not exhibit this property.

The Fc domain of adalimumab does not directly contribute to its inhibitory effects on osteoclast function, as demonstrated by F(ab’)2 fragments showing equivalent activity. However, the absence of an Fc domain in etanercept means it cannot induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), mechanisms available to monoclonal antibodies.

Etanercept:TNF complexes are cleared less efficiently by human osteoclasts than adalimumab:TNF complexes. While monomeric etanercept and adalimumab bind similarly to Fc-gamma receptors, their complexes with TNF show markedly different behavior. Adalimumab:TNF complexes bind to low affinity FcγR, particularly FcγRII, enabling efficient internalization by osteoclast precursors. In contrast, etanercept:TNF complexes maintain a monomeric binding profile without increased binding to low affinity FcγR, resulting in reduced internalization and clearance.

In proteomic studies, adalimumab:TNF complexes restore the protein expression profile of TNF-activated osteoclasts to that of RANKL alone, whereas etanercept:TNF complexes produce an intermediate profile. Adalimumab more effectively reduces TNF-induced protein levels (69% of upregulated proteins) compared to etanercept (20%). These differences extend to specific pro-osteoclastogenic factors: only adalimumab:TNF complexes reduce IGFBP2 and ICAM-1 to RANKL levels.

Synthesis

The differential efficacy of etanercept across disease contexts—effective in rheumatoid arthritis but failing to induce clinical response in Crohn’s disease—can be explained by specific mechanistic properties that distinguish it from monoclonal antibody TNF antagonists. Three key mechanistic features account for these divergent therapeutic outcomes.

First, etanercept’s 1:1 binding stoichiometry and formation of unstable complexes with soluble TNF means that not all TNF binding sites are blocked. In rheumatoid arthritis, where soluble TNF predominates in the joint space, even partial neutralization may provide therapeutic benefit. However, in inflammatory bowel disease, where membrane-bound TNF plays a critical role in pathogenesis, etanercept’s inability to effectively engage with this form becomes limiting. The preferential binding of etanercept to soluble TNF-α rather than membrane-bound TNF further explains its limited efficacy in conditions where the latter is pathogenically important.

Second, etanercept’s failure to induce apoptosis in mucosal lymphocytes and its inability to trigger TGF-β production through reverse signaling represent critical mechanistic deficiencies in the inflammatory bowel disease context. While infliximab induces apoptosis, IL-10 expression, and TGF-β production through membrane-bound TNF reverse signaling, etanercept lacks these immunosuppressive effects. The absence of an Fc domain precludes antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, mechanisms that may contribute to therapeutic efficacy in inflammatory bowel disease but are less essential in rheumatoid arthritis.

Third, the differential clearance and internalization kinetics of etanercept:TNF complexes explain dose-response relationships. Etanercept has approximately 13-fold higher clearance than adalimumab or infliximab, potentially requiring higher or more frequent dosing to achieve equivalent TNF neutralization. In osteoclast models, etanercept:TNF complexes are internalized less efficiently than adalimumab:TNF complexes through Fc-gamma receptors, leading to prolonged exposure of cells to locally available TNF. This reduced clearance efficiency may be therapeutically limiting in tissue compartments where efficient removal of TNF is critical, such as the intestinal mucosa in Crohn’s disease, while being adequate in the synovial environment of rheumatoid arthritis.

The bone protection observed in rheumatoid arthritis patients treated with etanercept appears sufficient despite mechanistic limitations because the drug successfully restricts TNF access to TNF-RI on osteoclast precursors, even if less effectively than adalimumab. The intermediate proteomic profile produced by etanercept:TNF treatment—between that of TNF alone and RANKL alone—still provides clinical benefit in the joint, where multiple pro-inflammatory factors operate and partial TNF neutralization contributes to overall disease control. In contrast, in inflammatory bowel disease, where membrane-bound TNF and reverse signaling appear particularly important, this intermediate level of pathway modulation falls below the therapeutic threshold.

These mechanistic distinctions do not represent flaws in etanercept’s design but rather reflect its origins as a soluble receptor construct versus a monoclonal antibody. The clinical implications are clear: etanercept is appropriately matched to diseases where soluble TNF predominates and where partial neutralization, without reverse signaling or apoptosis induction, suffices for therapeutic benefit. Conversely, in inflammatory bowel disease and potentially other conditions requiring membrane-bound TNF engagement, etanercept’s mechanistic profile predicts limited efficacy, as confirmed by clinical trial results.

References

K. Papp, E. Keystone, N. Shear\ (2007).Mechanism of Action, Pharmacokinetics, and Drug Interactions of Etanercept in Dermatology. Journal of Cutaneous Medicine and Surgery

U. Billmeier, W. Dieterich, M. Neurath, R. Atreya\ (2016).Molecular mechanism of action of anti-tumor necrosis factor antibodies in inflammatory bowel diseases. World Journal of Gastroenterology

D. Furst, R. Wallis, M. Broder, D. Beenhouwer\ (2006).Tumor necrosis factor antagonists: different kinetics and/or mechanisms of action may explain differences in the risk for developing granulomatous infection. Seminars in Arthritis & Rheumatism

Z. Szondy, Anna Pallai\ (2017).Transmembrane TNF‐alpha reverse signaling leading to TGF‐beta production is selectively activated by TNF targeting molecules: Therapeutic implications. Pharmacological Research

A. Tuttolomondo, R. Pecoraro, A. Pinto\ (2014).Studies of selective TNF inhibitors in the treatment of brain injury from stroke and trauma: a review of the evidence to date. Drug Design, Development and Therapy

Heejin Lim, Sang Hyung Lee, Hyun Tae Lee, Jee Un Lee, J. Son, and 2 more\ (2018).Structural Biology of the TNFα Antagonists Used in the Treatment of Rheumatoid Arthritis. International Journal of Molecular Sciences

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

B. Harvey, J. Cohen-Solal, Z. Kaymakcalan\ (2018).SAT0058 Adalimumab:tnf complexes are cleared more efficiently by human osteoclasts than those with etanercept through fcg-receptor binding and internalisation. Saturday, 16 JUNE 2018

B. Harvey, L. Jin, J. Maull, C. Hu, Y. Tian, and 1 more\ (2020).FRI0367 ADALIMUMAB IS MORE EFFECTIVE THAN ETANERCEPT AT PREVENTING TNF-ENHANCED OSTEOCLAST DEVELOPMENT THROUGH DOWNREGULATION OF PRO-OSTEOCLASTOGENIC FACTORS ICAM-1 AND IGFBP2 AND UPREGULATION OF ANTI-OSTEOCLASTOGENIC FACTOR FABP4. Annals of the Rheumatic Diseases

B. Harvey, C. Hu, D. Wang, Y. Tian, Z. Kaymakcalan\ (2016).AB0063 Complex of Adalimumab and TNF Induces A Divergent Proteomic Profile in TNF-Stimulated Human Osteoclast Precursors To That Resembling A Monocytic Cell. Annals of the Rheumatic Diseases

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Molecular mechanism of action of anti-tumor necrosis factor antibodies in inflammatory bowel diseases

U. Billmeier, W. Dieterich, M. Neurath, R. Atreya

World Journal of Gastroenterology·

2016·

191 citations

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Study Design

- Type of study: In vitro, mechanistic - Cell types or model systems used: Peripheral blood mononuclear cells (PBMCs), cell lines stably expressing mTNF - Duration of experiments: Not mentioned - Sample sizes or replicates: Not mentioned

Etanercept TNF-Binding Mechanism

- Binding stoichiometry: 1:1 ratio - Binding affinity/avidity: Similar affinity to golimumab, greater avidity than adalimumab and infliximab - Specific TNF forms bound: Preferentially binds to soluble TNF-α (sTNF) - Binding kinetics: Forms relatively unstable complexes with sTNF - Structural aspects: Chimeric fusion protein consisting of the p75 part of TNFR2 and a human IgG1 Fc domain - Unique binding characteristics: Higher avidity for sTNF compared to adalimumab and infliximab; unable to induce TGFβ in human macrophages

Downstream Signaling Pathways

- Activation of TNFR1 leads to intracellular signaling cascades involving apoptosis, cell proliferation, and cytokine secretion. - NF-κB is activated, leading to transcriptional upregulation of genes like IL8, IL1, IL6, COX2, and TNF. - TNFR1 can activate a Caspase8 dependent pathway resulting in apoptosis. - TNFR2 pathway is involved in proliferation, migration, and cytokine production, and can mediate reverse signaling. - Etanercept forms unstable complexes with sTNF, contrasting with other anti-TNF agents. - Etanercept fails to induce TGFβ in human macrophages. - Etanercept's binding does not fully engage signaling pathways as effectively as other anti-TNF agents.

Cellular Effects

- Cell viability/apoptosis effects: Etanercept does not induce apoptosis in peripheral blood lymphocytes or lamina propria T cells. - Cell differentiation impacts: Not mentioned. - Inflammatory mediator production changes: Etanercept partially inhibits IL1 beta release. - Cell migration/adhesion effects: Not mentioned. - Tissue-specific responses: Not mentioned. - Immune cell function modifications: Etanercept does not affect IL10 or IL12 expression; it fails to induce TGFβ.

Reverse Signaling Effects

Etanercept does not trigger reverse signaling in the same way as infliximab and golimumab. It fails to induce TGF-β production in human macrophages and does not activate downstream pathways such as IL-10 expression, ROS accumulation, and apoptosis. This suggests that etanercept does not effectively engage in reverse signaling effects associated with transmembrane TNF-α.

Comparative Mechanisms

- Mechanistic differences in TNF binding: Etanercept forms unstable complexes with sTNF and binds in a 1:1 ratio, not blocking all binding sites. - Differences in downstream signaling activation: Etanercept does not induce TGFβ in human macrophages, which is associated with immune suppression. - Comparative cellular effects: Etanercept does not induce apoptosis in lymphocytes from CD patients and lacks an Fc domain, preventing ADCC and CDC. - Differential pathway activation/inhibition: Etanercept has higher affinity and avidity for sTNF but fails to induce clinical response in Crohn’s disease. - Unique properties specific to etanercept vs. monoclonal antibodies: Etanercept is a chimeric fusion protein with a unique structure and lacks an Fc domain.

Experimental Methods

- Binding assays: Surface plasmon resonance to measure affinity to soluble TNF. - Signaling pathway analysis: Formation of unstable complexes with sTNF. - Proteomics/genomics approaches: Gene regulation analysis using intestinal biopsies. - Cell culture conditions and treatments: In vitro experiments with intestinal T cell cultures and PBMCs. - Analytical methods for measuring outcomes: CDC assays for cell death; ADCC comparison. - Controls and comparators: Comparison with infliximab, adalimumab, and certolizumab pegol.

Key Mechanistic Findings

- Primary mechanistic insights: Etanercept forms unstable complexes with sTNF and binds in a 1:1 ratio, suggesting incomplete blockade of TNF activity. - Novel discoveries about etanercept's action: Etanercept fails to induce TGFβ in human macrophages and is incapable of inducing apoptosis in lymphocytes. - Clinical implications of mechanistic findings: Etanercept is ineffective in treating Crohn's disease due to its inability to effectively neutralize mTNF. - Limitations or gaps in mechanistic understanding: The paper highlights the limitations of etanercept's mechanism, particularly its inability to effectively neutralize mTNF. - Authors' interpretations of mechanism-effect relationships: The authors suggest that etanercept's failure to induce clinical response in Crohn's disease is due to its mechanistic limitations, particularly its inability to form stable complexes with mTNF.

Anti-tumor necrosis factor (TNF) antibodies are successfully used in the therapy of inflammatory bowel diseases (IBD). However, the molecular mechanism of action of these agents is still a matter of debate. Apart from neutralization of TNF, influence on the intestinal barrier function, induction of apoptosis in mucosal immune cells, formation of regulatory macrophages as well as other immune modulating properties have been discussed as central features. Nevertheless, clinically effective anti-TNF antibodies were shown to differ in their mode-of-action in vivo and in vitro. Furthermore, the anti-TNF agent etanercept is effective in the treatment of rheumatoid arthritis but failed to induce clinical response in Crohn’s disease patients, suggesting different contributions of TNF in the pathogenesis of these inflammatory diseases. In the following, we will review different aspects regarding the mechanism of action of anti-TNF agents in general and analyze comparatively different effects of each anti-TNF agent such as TNF neutralization, modulation of the immune system, reverse signaling and induction of apoptosis. We discuss the relevance of the membrane-bound form of TNF compared to the soluble form for the immunopathogenesis of IBD. Furthermore, we review reports that could lead to personalized medicine approaches regarding treatment with anti-TNF antibodies in chronic intestinal inflammation, by predicting response to therapy.

INTRODUCTION

Inflammatory bowel diseases (IBD) are chronic infla mmatory disorders mainly affecting the gut. The two main forms are Crohn's disease (CD) and ulcerative colitis (UC) that differ in several aspects, e.g. regarding the location and distribution of inflammation and in the mucosal cell populations involved in the immune reaction. CD is a segmental, transmural disorder that can affect the whole gastrointestinal tract and type 1 T helper cells have been associated to be involved in the pathogenesis [1] . In contrast, UC is characterized by continuous inflammation of the colon, and a modified Th2 cytokine profile has been described [2] . Additionally, the recently described Th17 cells have been reported to be present in both CD and UC [3] . However, the precise etiology of IBD is still a matter of debate but there is general consensus that genetic predisposition, environmental factors and immunological dysfunction of tolerance against the intestinal microflora are involved in the immunopathogenesis [4] . In this regard, mucosal CD4+ T cells as key mediators in driving immune responses are critically involved in the patho genesis of IBD and in accordance, a high number of infiltrating T lymphocytes can be found in both CD and UC in the gut [5] . Furthermore, recent studies strongly suggest that the proinflammatory cytokine tumor necrosis factor (TNF) is one of the major pathogenic cytokines involved in the pathogenesis of IBD as elevated levels of TNF are present in the serum of both UC and CD patients [6] . In addition, an elevated number of TNFsecreting cells in the inflamed mucosa of IBD patients has been repeatedly reported [79] . Herein, lamina propria mononuclear cells (LPMCs) isolated from colonic biopsies from IBD patients spontaneously produced increased amounts of TNF which correlated with the degree of tissue involvement and mucosal inflammation [10] , strengthening the importance of TNF in the inflamed gut.

In accordance, application of antibodies targeting TNF in IBD patients have been shown to induce clinical response in up to 60% of CD patients, and inducing longterm maintenance of remission in a large amount of patients [1113] . Comparable results have also been described for the therapy of UC patients [14, 15] . Nevertheless, the molecular mechanism of action of TNF agents is still discussed and not all commercially available antiTNF antagonists are effective in the therapy of IBD patients. Regarding the mechanism of action, TNF neutralization, diverse effects on the immune system, outsidetoinside signaling and importantly, induction of direct or indirect apoptosis have been suggested. Furthermore, first studies focusing on successful prediction of clinical response have been conducted, either via expression of various biomarkers [16,17] or by endoscopic in vivo molecular imaging [18] . In this review, TNF biology and different antiTNF antibodies will be assessed, and the results of different aspects of the biological function of anti TNF agents for the treatment of IBD will be discussed.

TNF BIOLOGY AND ITS SIGNALING PATHWAY

TNF is a crucial mediator in driving inflammatory processes in the gut. It is produced by a variety of mucosal cells, mainly macrophages and T cells, as a preform on the plasma membrane [19] . In addition, Paneth cells in CD affected segments of the terminal ileum were shown to strongly express TNF RNA in contrast to Paneth cells in normal tissue, indicating an induction under pathogenic conditions [20] . The transmembrane precursor form (mTNF), a homotrimer of 26 kDa subunits, is cleaved by the matrixmetallo proteinase TNF alpha converting enzyme (TACE/ Adam17) into a soluble form (sTNF, a homotrimer of 17 kDa monomers) [2123] . The expression of mTNF by CD14+ macrophages has been reported to be relevant in IBD [24] (Figure 1 ).

Both forms of TNF are biologically active and signal through two distinct receptors that differ in molecule mass: the 55kDa TNFR1 (TNFRSF1A/ CD120a) and the 75kDa TNFR2 (TNFRSF1B/CD120b) glycoproteins [25] . TNFR2 is mainly expressed on lymphocytes and endothelial cells, whereas TNFR1 is ubiquitously expressed and possesses an intracellular death domain [26] . TNF and its receptors are crucially involved in the pathogenesis of IBD. For example, elevated levels of the soluble form of TNFR1 and TNFR2 have been detected in both CD and UC patients and their expression correlated with disease activity [27] . Signal transduction of the membranebound form of TNF can be transmitted through both TNFR1 and TNFR2, whereas sTNF mainly signals through TNFR1. Binding affinity studies revealed that sTNF preferentially binds to TNFR1 with higher affinity [28] . In contrast, TNFR2 is mainly activated by mTNF [29] . Activation of TNFR1 by TNF induces an intracellular signaling cascade with pleiotropic effects involving apoptosis, cell proliferation or cytokine secretion. Activation of the nuclear factor kappa B (NFκB) following stimulation of TNFR1 results in translocation to the nucleus and transcriptional upregulation of several genes such as IL8, IL1, IL6, COX2 and TNF [21] . Alternatively, TNFR1 can activate a Caspase8 dependent signaling pathway via FADD resulting in apoptosis. The TNFR2 pathway does not contain a death domain and its stimulation can result in proliferation, migration and cytokine production such as IL1 and IL6. Furthermore, binding of mTNF to TNFR2 not only activates an intracellular signaling pathway, but can also result in reverse signaling within the TNFexpressing cell [30] which will be later discussed in detail. The role of the receptors of TNF in the pathogenesis of IBD remains only partly understood. A study using a colitis mouse model suggests that both TNFR1 and TNFR2 have protective functions in intestinal inflammation [31] . Herein, intestinal inflammation was provoked by oral application of dextran sulfate in mice deficient for TNFR1 or TNFR2 as well as wildtype controls. TNFR1 or TNFR2 ablation resulted in exacerbation of colitis, possibly due to increased apoptosis of colonic epithelial cells [31] . However, in other studies a central role of TNFR2 in mucosal inflammation has been proposed. For example, mutations in the gene of TNFR2 have been linked with IBD [32,33] , suggesting that this polymorphism could increase the disease risk. In accordance, its overexpression drives inflammation in a transgenic mouse stain overexpressing human TNFR2, causing a severe multiorgan inflammatory syndrome mainly affecting liver, pancreas, kidney and lung [34] . Regarding IBD, TNFR2 expression was found to be upregulated on lamina propria and peripheral blood T cells in patients with CD [35] . In accordance, TNFR2 was found to promote experimental colitis. Herein, T cells overexpressing TNFR2 were transferred into SCID mice that do not express T and B cells. In comparison to SCID mice that received wildtype T cells, transfer of T cells overexpressing TNFR2 resulted in more severe colitis and enhanced expression of T helper cells type 1 [35] , underlining the importance of TNFR2 in intestinal inflammation. It could moreover be shown that activation of mucosal TNFR2 expressing CD4+ T cells by mTNF expressing CD14+ led to heightened resistance of the intestinal lymphocytes to apoptosis, resulting in perpetuation of chronic intestinal infla mmation in IBD [24] .

Infliximab

In 1998, infliximab (Remicade ® ) was the first antibody targeting TNF to be approved in the United States for the treatment of CD and later for the treatment of UC. It is a chimeric monoclonal antibody with 25% murine and 75% human sequences. Infliximab therapy is intravenously initiated at weeks 0, 2 and 6 and then applied every 8 wk for maintenance of remission. The starting concentration for both CD and UC is 5 mg/kg [6] .

CT-P13

The infliximab biosimilar CTP13 (Remsima ® , Inflectra ® ) was approved in Europe for the treatment of both CD and UC based on a comprehensive nonclinical comparability exercise and extrapolation of clinical data from two studies with rheumatoid arthritis patients. The biosimilar product is highly similar to its originator biological drug infliximab and is therefore clinically used in the same way.

Adalimumab

In contrast to infliximab, adalimumab (Humira ® ) is a monoclonal human antibody produced by CHO cells. It is approved for the treatment of both CD and UC. Adalimumab is administered subcutaneously and can be injected by the patient himself. At therapy initiation, adalimumab is administered at 160 mg and then 80 mg two weeks later. For maintenance of remission adalimumab is administered every 2 wk at 40 mg.

Golimumab

In 2013 golimumab (Simponi ® ), a fully human mono subcutaneously applied twice per week [39] . Etanercept is therefore not approved for treatment of CD.

ANTIBODIES

Although antiTNF antibodies are approved for the therapy of IBD and other inflammatory diseases for many years, the mechanism of action is still a matter of debate. Initially, it was suggested that antiTNF agents inactivate the proinflammatory cytokine TNF by direct neutralization, thus resulting in suppression of inflammation. Given the complexity of TNF signaling, a lot of studies indicate that antiTNF antibodies exert more complex functions beyond simple blockade. Furthermore, functional characterization of etanercept and other antiTNF agents can elucidate the signaling pathways that are important in IBD in contrast to other autoimmune diseases such as rheumatoid arthritis. In the following part we will list several described effects of antiTNF antibody therapy that are suggested to be relevant in IBD (see also Figure 2 ).

TNF NEUTRALIZATION

Although all antiTNF agents have the same target, the affinity of the antibodies to TNF has been reported not clonal antibody was approved for the treatment of UC [36] . Like adalimumab, it is administered sub cutaneously. The induction dose is 200 mg and then 100 mg two weeks later. Maintenance of remission is achieved by 50 mg (< 80 kg body weight) and 100 mg (≥ 80 kg bodyweight) every 4 wk.

Certolizumab pegol

Certolizumab pegol (Cimzia ® ), a PEGylated humanized Fab' fragment is approved for the treatment of CD in the United States and Switzerland [37] but not by the European Medicines Agency. In contrast to the other antiTNF antibodies, this agent does not possess a Fc fragment and correspondingly, is not recognized by Fc receptors. The subcutaneous administration is performed at week 0, 2 and 4 with 400 mg. For maintenance of remission, certolizumab pegol is given every 4 wk in a dose of 400 mg.

Etanercept

Etanercept (Enbrel ® ) is a chimeric fusion protein that consists of the p75 part of TNFR2 and a human IgG1 Fc domain. Importantly, in combination with methotrexate, etanercept has similar efficacy in the therapy of rheumatoid arthritis as infliximab and adalimumab [38] . It nevertheless failed to be effective in a clinical study with CD patients, when 25 mg were

to be equal. An overview of respective affinities and avidities of antiTNF agents is given in Tables 1 and 2 .

In TNF bioassays, infliximab, etanercept, adalimumab, certolizumab pegol and golimumab all neutralize sTNF [23] . However, using the method of surface plasmon resonance, golimumab was shown to bind to soluble TNF with a similar affinity as etanercept, but with greater affinity than infliximab and adalimumab, the later showing the weakest affinity in the used setting [40] . However, affinity of adalimumab and infliximab were reported to be similar although less compared to etanercept in another study [41] . Regarding avidity, etanercept was shown to bind to sTNF with a 10 to 20fold greater avidity as compared to adalimumab or infliximab [41] . For mTNF, affinity of antiTNF antibodies has been reported to be lower than to sTNF [40,41] . Using mTNF transfected cells, binding affinities of infliximab, adalimumab and etanercept were shown to be similar [41] . In contrast, affinities for golimumab and infliximab to another mTNFexpressing cell line were comparable and greater than that of adalimumab, whereas all three antiTNF agents had significantly greater affinity than etanercept [40] . Furthermore, in other studies binding of etanercept to mTNF could not be detected, perhaps due to the fact that peripheral blood mononuclear cells (PBMCs) and not cell lines stably expressing mTNF were examined [42,43] , arising the question to what extent the binding of etanercept to mTNF expressing cell lines represents the situation in vivo. Furthermore and in discrepancy to the above mentioned study, etanercept was shown to form relatively unstable complexes with sTNF, resulting in fast release of dissociated TNF in contrast to infliximab that bound to both sTNF and mTNF by forming stable complexes with high stability [44] . However, interpretations of all presented data have to take into account the different cells, methods and techniques used for respective assessment of affinity, which could have a profound effect on the data obtained. Importantly, another aspect is the variation of the different antiTNF agents in regard to crosslinking towards mTNF. Herein, it has been reported that up to three infliximab molecules can bind to each TNF homotrimer. In contrast, etanercept was shown to bind to TNF trimer in a 1:1 ratio, suggesting that not all binding sides are blocked [44] . This aspect together with the fact that infliximab forms more stable complexes with mTNF [44] is a possible explanation why infliximab is effective in the therapy of IBD, whereas etaner cept failed to show any clinical efficacy in a trial with Crohn's disease patients and is therefore not approved for IBD treatment. Recent findings indicate that the neutralization of mTNF rather than sTNF is crucial in the therapy of IBD. For example, in a mouse model of IBD the neutralization of sTNF alone was ineffective, in contrast to antiTNF antibodies that block both sTNF and mTNF, indicating that neutralization of mTNF is crucial in the therapy of IBD [45] . The pivotal role of mTNF was further demonstrated in an experimental colitis model by transferring CD4+ T cells into RAG2 deficient mice [46] . Herein, by transferring TNF mutant T cells, disease activity was compared in absence of TNF, in absence of sTNF and in presence of both sTNF and mTNF. Whereas the expression of TNF was essential for the induction of colitis, the absence of sTNF did not protect the mice from intestinal inflammation [46] , clearly underlining the pathogenic role of mTNF in the pathogenesis of mucosal inflammation in the intestine.

Cytokines

Cytokines as mediators of inflammation are in imbalance in IBD. On the one hand proinflammatory cytokine levels such as TNF, IL13, IL17 or IFNγ [13,6] are elevated and drive the massive tissue damage in the gut. On the other hand, antiinflammatory cytokines that usually regulate overwhelming immune reactions are supposed to be also involved in the pathogenesis of IBD. For example, mice deficient of IL10 spontaneously develop a chronic intestinal inflammation, which is dependent on the influence of the microflora [47] . Interestingly, the enterocolitis exacerbates in mice deficient for both IL10 and TNF, indicating that TNF has some protective effects in this context [48] . Crucial mediators of inflammation in the gut are neutrophils and granulocytes that accumulate in inflamed areas and secrete proinflammatory cytokines, chemokines and recruit effector cells to the side of inflammation. In this setting, Agnholt et al [49] reported that GMCSF, a growth factor for granulocytes and neutrophils, was decreased in CD patients after application of infliximab. In accordance, also reduced peripheral blood neutrophils and diminished infiltration of polymorphonuclear cells in the lamina propria were observed after antiTNF application. Furthermore, in vitro experiments using an intestinal T cell culture system showed that GM CSF secretion by T cells was inhibited by infliximab, suggesting direct effects of infliximab on T cells. Apart from GMCSF, the release of the proinflammatory cytokine IL1 beta after stimulation of human mono cytes with lipopolysaccharide (LPS) was inhibited by antiTNF agents. Herein, certolizumab pegol was the most potent inhibitor. Infliximab and adalimumab were also able to inhibit IL1 beta release at higher concentrations whereas etanercept only partially prevented IL1 beta release [50] . Furthermore, in vitro analysis using PBMCs from patients with UC showed that incubation of the cells with infliximab inhibited secretion of the proinflammatory cytokines IFNγ, IL13, IL17A and TNF [51] , cytokines reported to be critically involved in the pathogenesis of IBD. In addition, infliximab reduced the expression of TNF, IL1, IL6 and IL8 by LPSstimulated monocytes in vitro [21,52] . In addition, the key Th1 cytokine IL12 and the antiinflammatory cytokine IL10 were described to be affected by antiTNF antibodies. Monocytes that were isolated from blood of healthy controls or CD patients expressed less IL10 or IL12 when they were incubated in the presence of adalimumab or infliximab, whereas etanercept had no effect in this setting [53] . Interestingly, a recent study analysed the regula tion of 14169 different genes by antiTNF antibodies and compared the changes in antiTNF responders versus nonresponders. Intestinal biopsies from CD patients were used and compared to controls [54] . They identified some genes that were regulated by antiTNF treatment, such as IL6, CD69, GPR183 and MMP9. Interestingly, the proinflammatory cytokine IL6 was downregulated by antiTNF agents but independently of clinical response to therapy. Of note, IL1 beta and IL17A were found to be significantly upregulated in patients with active CD and patients refractory to antiTNF, in accordance with an enhanced Th17 cell population in IBD [3] , thereby representing possible targets in antiTNF refractory patients. In contrast to this study, Katz el al [55] found IL17 levels not to be affected by antiTNF therapy in CD patients but showed IL2 to be significantly decreased during anti TNF therapy. These contradictory findings may reflect biological differences between gene expression and protein expression.

T helper cell subsets

T cells are critically involved in the pathogenesis of IBD and therefore, modulation of immune responses by influencing the presence of T helper cell subsets is suggested to be an important effect of antiTNF agents. Response and clinical remission could be the result of either a reduction in proinflammatory T cell subsets such as Th1, Th2 or Th17 cells or an enhanced presence of antiinflammatory T helper cells, or both. In this regard, several studies investigated the number of pro and antiinflammatory T helper cell subsets before and after antiTNF therapy. The influence of infliximab in vitro on isolated T cells from the peripheral blood of UC patients was examined by Dahlén et al [51] . The T cell activation marker CD25 was reduced on both CD4+ and CD8+ blood T cells and their proliferation was inhibited after incubation with infliximab. In accordance, the levels of proinflammatory cytokines such as IFNγ, IL13, IL17A, TNF and granzyme A were inhibited. The authors also investigated effects of infliximab on regulatory T cell populations; however, the results regarding CD4+ Foxp3+ T cells were ambiguous [51] . Importantly, effects of infliximab on blood or mucosal T cells were shown not to be identical. Herein, Li et al [56] described that infliximab therapy resulted in an increase in CD4+CD25+Foxp3+ and CD4+CD25 Foxp3+ regulatory T cell populations in the blood of both UC and CD patients; in contrast, Foxp3 mRNA and protein were downregulated in the mucosa, possibly reflecting increased apoptosis rates or down regulated T cell activation in the intestine during therapy. Other studies described general reduction of T cells in the mucosa after infliximab therapy for both CD [57] and UC patients [58] . A direct effect of adalimumab on T cell populations could be elegantly demonstrated by Maggi et al [59] . Local injection of adalimumab along perianal fistulas in CD patients resulted in decrease of infiltrating CD4+ CD161+ T cells whereas no systemic effects on circulating T cells could be detected [59] . In addition, infliximab has been shown not only to suppress CD4 T cell activation, but also to down regulate IL21 expression by mucosal CD4+ T cells and consequently, inhibit Th17 differentiation. Using intestinal biopsies of CD patients before and after infliximab therapy, the authors could clearly show that intestinal mucosal healing during therapy was due to downregulation of mucosal infiltration of the highly proinflammatory Th17 cells through diminished IL21 expression, a cytokine that is pivotal to Th17 cell differentiation [60] . In summary, these studies clearly indicate that effects of antiTNF antibodies on T cells are complex and differ between peripheral and mucosal T cells, as well as in vivo and in vitro.

Epithelial cells, angiogenesis and immunosuppressive macrophages

In addition to shifts in cell populations, effects of anti Billmeier U et al . Anti-TNF antibodies in IBD TNF antibodies on the epithelial barrier function have been also examined. For example, adalimumab has been shown to prevent barrier dysfunction induced by TNF in vitro using the intestinal epithelial cell lines Caco2 and T84 [61] . Furthermore, a role of infliximab in restoring epithelial barrier dysfunction in CD has been described. Herein, downregulation of epithelial apoptosis was noticed in 10 of 11 patients undergoing therapy with infliximab [62] . Additionally, effects of antiTNF antibodies on the mucosal microcirculation have been reported. In this context, infliximab was shown to downregulate CD40 expression by intestinal microvessels in vivo in the mucosa of CD patients on the one hand and to reduce the levels of sCD40L in the circulation on the other hand, indicating an inhibition of vascular inflammation in the gut [63] . In addition, infliximab downregulated mucosal angiogenesis in CD patients [64] . A direct effect of infliximab on myofibroblasts has been described by Di Sabatino et al [65] . Myofibroblasts isolated from CD patients expressed more mTNF than control myofibroblasts and infliximab significantly augmented the migration ability from CD myofibroblasts in a tissue inhibitor of metalloproteinases (TIMP)1dependent manner, suggesting another modeofaction of antiTNF anti bodies that results in enhanced mucosal healing. However, the TIMP1 production was also increased by adalimumab and etanercept, so it is still unclear if this effect is relevant for induction of clinical remission in CD. Interestingly, the induction of immunosuppressive macrophages has been suggested as mechanism of action of antiTNF antibodies [42] . Therefore, the influence of infliximab, adalimumab, certolizumab pegol and etanercept was investigated in PBMCs from healthy controls. The T cell proliferation was diminished by infliximab and adalimumab in a mixed lymphocyte reaction, whereas certolizumab pegol and etanercept had no effect. The inhibition of proliferation was driven by immunosuppressive CD14+ macrophages that secreted large amounts of the antiinflammatory cytokine IL10 and were activated by the Fc region of the antibodies [42] . The same group later showed that regulatory macrophages were induced in IBD patients with mucosal healing upon infliximab treatment and functionally, these macrophages had the ability to induce wound healing in vitro [66] . In summary, several effects of antiTNF antibodies on the immune system have been described. The reduction of proinflammatory T cell subsets on the one hand and the induction of regulatory T cells on the other hand clearly contributes to resolution of inflammation. In addition, the induction of regulatory macrophages [42] and effects on mucosal healing [61,62,65] have been suggested.

OUTSIDE-TO-INSIDE SIGNALLING

As mentioned before, activation of the membrane bound form of TNF can result in bidirectional signalling.

The function of mTNF not only as a ligand but also as a receptor is called "outsidetoinside signal" or reverse signalling. Reverse signalling by mTNF on monocytes/ macrophages confers resistance to bacterial LPS [67] , suggesting that this pathway represents a silencing signal. A very recent paper demonstrated that the immunesuppressive effect of mTNF following LPS stimulation in macrophages was mediated by TGFβ via activation of the MAPK kinase, interestingly this effect was mediated by infliximab and golimumab whereas etanercept failed to induce TGFβ in human macrophages [68] . Functional studies showed that LPS resistance downstream of mTNF is mediated through protein kinaseC dependent and independent pathways in monocytic cells [67] . In addition, enhanced secretion of IL2 and IFNγ [69] and an upregulation of Eselectin in T cells have been reported [70] . Furthermore, apoptosis induction as consequence of reverse signalling has also been proposed in CD patients [43,71] . A study by Mitoma et al [72] shed light on the reverse signalling pathway induced by infliximab and etanercept. Herein, human Jurkat T cells were stably transfected with a wildtype and a mutant form of mTNF. Eselectin expression was induced by both agents, but only infliximab induced the expression of the antiinflammatory cytokine IL10, activation of ROS accumulation and apoptosis by upregulation of Bax and Bak [72] . Interestingly, their finding that infliximab further induced G0/G1 cell cycle arrest elucidated another mechanism of action of anti TNF antibodies. Later the induction of cell cycle arrest was shown to be mediated by adalimumab as well [73] .

In the context of reverse signalling, a downregulation of the growth and differentiation factor1 (GDF1) mediated by infliximab and certolizumab pegol has been described [74] . Using gene expression assays, GDF1 was found to be upregulated in inflamed tissue from CD patients, and downregulated after infliximab treatment in clinical responders. Furthermore, GDF1 was shown to act as proinflammatory mediator by IL6 and STAT3 induction [74] , indicating that a down regulation of proinflammatory effectors can also be a consequence of reverse signalling by antiTNF agents in contrast to immunesuppressive effects.

APOPTOSIS

The programmed cell death of immune cells is a fundamental mechanism of resolution of inflammation. Apoptosis induction by antiTNF antibodies has therefore been addressed by several groups. The induction of apoptosis can either be direct or indirect as side effect of TNF signaling. Furthermore, apoptosis can be induced by the Fc region of antiTNF antibodies involving the complement or natural killer cells or can be a consequence of the activation of TNF itself by antibody binding. An overview of the apoptosis inducing capabilities of the different antiTNF antibodies is given in Table 3 .

ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY AND COMPLEMENT-DEPENDENT CYTOTOXICITY

Antibodydependent cellular cytotoxicity (ADCC) is a mechanism of the adapted immune system in order to kill antibodytagged target cells, for example infected cells. Therefore, after binding of an antibody to its target cell, the Fc domain is recognized by the Fc receptor of effector immune cells, typically natural killer cells. The natural killer cell then releases cytotoxic proteins such as perforins and granzymes that subsequently results in lysis of the target cell. ADCC is a mechanism of action of antiTNF antibodies that possess a Fc domain. A recent study indicated that ADCC measured as cell lysis of coincubation of mTNFexpressing CHO cells and PBMCs could be induced by adalimumab in up to 76% of cells in contrast to a control isotype that caused ADCC in less than 5% of the cells [75] . In a comparative study, infliximab and adalimumab were shown to induce ADCC in a similar way and more potent as etanercept whereas certolizumab pegol did not show any effect due the fact that the later agent does not possess the Fc domain [50] . Apart from ADCC, binding of antibodies to a target cells can result in complement activation. The com plement consists of several different proteins that act as a cascade which finally leads to the formation of a membrane attacking complex, inducing a pore within the cell and subsequent cell death. In a CDC assay, 10 μg/mL adalimumab were shown to induce 90.6% cell death in the presence of heatinactivated human serum [75] . Comparing different agents, infliximab and adalimumab had the same capacity in inducing CDC dependent cell death and were more effective than etanercept and as expected, certolizumab pegol did not show any effect [50] , which was in accordance to other studies [41,73] . Of note, CDC was induced in mTNF transfected cells by adalimumab and infliximab, but not in activated human PBMCs [41] , raising the question if cell lines overexpressing mTNF reflect the situation in vivo. In a functional study, affinity of infliximab and adalimumab to FcRγII and FcRγIII receptors was found to be low but significantly increased in the presence of exogenous TNF [76] . Similarly, both antibodies bound to the complement protein C1q in the presence of TNF. In both settings, etanercept binding was shown to be low. The more recently approved antiTNF antibody golimumab induced both CDC and ADCC to a similar extent as infliximab and adalimumab [77] . Fcmediated apoptosis has been shown to be most potently induced by infliximab and adalimumab. However, the fact that certolizumab pegol is effective in CD despite its incapacity to induce ADCC or CDC strongly suggests that Fcmediated apoptosis by anti TNF antibodies is not a central mechanism of action.

APOPTOSIS OF INFLAMMATORY IMMUNE CELLS

Apoptosis of inflammatory immune cells as consequence of antiTNF treatment has been discussed as a major mechanism of action explaining the fast onset of therapeutic effects upon successful therapy. Thus, the induced cell death of monocytes/macrophages or T cells has been analyzed. Regarding monocytes, in a comparative study both adalimumab and infliximab induced apoptosis in cultured monocytes rapidly in a caspasedependent way whereas etanercept did not [53] . In addition, apoptosis induction via transmembrane TNF in a human monocytic cell line by adalimumab and infliximab could be detected in a chimeric mouse model and furthermore, apoptosis could be abrogated by in vivo treatment with a pancaspase inhibitor, indicating a caspasedependent mechanism of action [78] . Infliximabinduced apoptosis of monocytes required the activation of members of the caspase family, such as caspase8, 9 and 3 in peripheral blood monocytes derived from CD patients [79] . Activation of caspase was induced independently from the CD95/ CD95L signaling pathway by release of mitochondrial cytochrome C, apparently triggered by Bax and Bak [79] .

The effect of antiTNF agents on lymphocytes was studied by Van den Brande et al [43] . They could show that etanercept, which is ineffective in the treatment of CD, in contrast to infliximab was incapable of inducing apoptosis in peripheral blood lymphocytes from healthy controls and lamina propria T cells from CD patients. Here, infliximab rapidly activated caspase 3 which possibly can also be a consequence of reverse signaling. Later the same authors demonstrated that this was also relevant in vivo. A rapid increase of apoptosis was detected in experimental colitis and importantly, also in CD patients where infliximab mediated apoptosis occurred within 24 h after starting the therapy [80] . In this in vivo molecular imaging study, patients underwent singlephoton emission computer tomography (SPECT) 24 h after infliximab infusion, and apoptosis was measured by radiolabeled annexinV uptake using scintigraphic imaging. A strong correlation of detected apoptosis signals and diseased regions was observed and furthermore, annexinV uptake was significantly higher in patients subsequently responding to infliximab therapy. In addition, in some patients LPMCs from biopsies were isolated and a marked increase in apoptosis of CD4 T cells after infliximab treatment was observed [80] .

Another study analyzing apoptotic markers such as Bcl2, Bax, Caspase3 and Fas in biopsies from CD patients before and after infliximab/adalimumab therapy showed that in LPMCs, a significant increase of active Caspase3 and an increase in the proapoptotic Bax/Bcl2 ratio could be detected upon antiTNF therapy, but no change of TNFR1 or Fas expression could be measured [81] . In recent studies of our group, another mechanism of action apart from direct induction of apoptosis by clinically effective antiTNF antibodies was identified. We showed that T cell apoptosis was only induced if mucosal T cells expressing TNFR2 were cocultivated with mTNFexpressing CD14+ macrophages [24] . Binding of mTNF to TNFR2 on T cells resulted in activation of TNFR2 dependent signaling pathways leading to TRAF2 and NFκB induction followed by heightened IL6 production, and subsequently resulting in T cell resistance to apoptosis, which leads to perpetuation of intestinal inflammation. This concept proposes an indirect apoptosis induction by neutralizing the mTNF binding side for TNFR2 by antiTNF antibody treatment, resulting in apoptosis of TNFR2 expressing T cells [24] .

PREDICTION OF CLINICAL RESPONSE

Given that approximately 30% of the patients fail to respond to antiTNF therapy (primary nonresponders), and up to 50% lose the response during therapy (secondary nonresponders) [82] , there is a clinical need for biomarkers allowing prediction of clinical response. Expression of biomarkers in blood or intestinal biopsies has been investigated in this regard. CD: Crohn's disease; UC: Ulcerative colitis; SPECT: Single-photon emission computer tomography.

Table 4 Prediction of response to anti-tumor necrosis factor therapy -possible biomarkers

revealed that modulation of TNFdependent genes has also been observed in nonresponsive patients, indicating biological activity of antiTNF independently of clinical response [54] . On the other hand, in this study IL1B and IL17A remained altered in nonresponders, suggesting these cytokines as relevant targets in this subgroup of patients [54] . A database search validated possible biomarkers in gene expression in patients with IBD under infliximab therapy. Of interest, datasets of PBMCs and biopsies were not comparable. Regarding intestinal biopsies, the genes IL13RA2, PTGS2 and WNT5A were shown to possibly predict the respon siveness to infliximab in IBD [16] .

Another study investigating UC patients before and after therapy with infliximab identified a high CAI, a negative antineutrophil cytoplasmatic auto antibody status and the IL23R genotype as predictors of response to therapy [17] . As apoptosis is a central mechanism of action of antiTNF antibodies, a predictive model based on an apoptotic pharmacological index (API) has been proposed in another study. Herein, response rates of infliximab in CD patients have been investigated in regard to polymorphisms of apoptosis genes (FAS ligand 843C/T; Fas 670G/A; Caspase9 93 C/T). Patients with an low API had the lowest response whereas patients with an high API displayed high response and remission rates [83] . In addition, high Creactive protein levels have been described as predictors for response to antiTNF therapy [84] . Gene array analyses of biopsies from UC patients taken before antiTNF treatment identified mainly five differentially expressed genes involved in adaptive immunity (osteoprotegerin, stanniocalcin1, prostaglandinendoperoxide synthase 2, IL13R alpha 2 and IL11) in patients later responding to infliximab as compared to patients that did not [85] . However, these promising biomarkers will have to be validated in larger cohorts of patients to verify if they serve as reliable markers for prediction of therapy in the clinic.

Another approach for prediction of response is based on biomarkers derived from the molecular mechanism of action of antiTNF antibodies and transition into corresponding in vivo molecular imaging modalities. As described earlier, apoptosis induction by infliximab was determined by SPECT imaging and detected a significantly higher apoptosis rate in patients later responding to infliximab [80] . The visualization of mTNF expressing cells by molecular imaging was the fundament of another recently published clinical trial. Based on the previous finding that the interaction of mTNF expressing cells and TNFR2 expressing T cells is targeted by antiTNF antibodies [24] , it was possible to predict clinical response to subsequent antiTNF therapy using GMPconform fluorescent antiTNF antibodies during endoscopy. Here, prior to initiation of antiTNF therapy, fluorescent antiTNF antibodies were topically applied in vivo to the inflamed mucosa in 25 CD patients and the number of mTNF positive mucosal cells were localized with molecular imaging using a confocal laser endomicroscope. It could be demonstrated that CD patients with high amounts of mTNF+ cells showed significantly higher response rates at week 12 (92%) upon subsequent antiTNF therapy as compared to patients with low amounts of mTNF+ cells (15%). Clinical response was defined as the reduction of the Crohn's Disease Activity Index > 100 points at week 12 after the initiation of the antiTNF therapy. A high number of mTNF positive cells also predicted sustained clinical response at week 52, decreased steroid use and high mucosal healing rates [18] . These findings represent a first step towards individualized medicine, making it possible to determine the most suitable biological therapy based on molecular level analysis thereby circumventing ineffective antibody therapy in IBD patients.

DISCUSSION AND FUTURE RESEARCH DIRECTIONS

Given the complexity of TNF signaling, the identification of the mechanism of action of antiTNF therapy remains challenging. In this review, several aspects of potential targets for antiTNF antibodies have been discussed. However, as reviewed here, contradictory results concerning the influence of antiTNF antibodies on cells in vivo and in vitro have been reported, which possibly reflect the different experimental conditions used in each study. Furthermore, although infliximab, adalimumab, golimumab, certolizumab pegol and etanercept target the same epitope, fundamental differences in their modeofaction in vivo have been demonstrated, beginning with the fact that etanercept is effective in the treatment of RA, but did not show therapeutic efficacy in the treatment of CD. However, in summary some conclusions can be drawn: First, as many IBD patients benefit from longterm remission induced by antiTNF therapy, additionally to TNF neutralization fundamental biological changes must be induced, especially in those patients that can stop the medication due to stable remission. Second, it is unclear if Fcdependent apoptosis such as ADCC and CDC by antiTNF agents is relevant in vivo in IBD. Certolizumab pegol is incapable of inducing ADCC and CDC due to its structure but is successfully used for the treatment of CD. Third, direct and indirect apoptosis induction of antiTNF agents and the modulation of the immune system possibly might be connected. For example, apoptosis induction could possibly directly influence the mucosal cytokine production [53] . Fourth, the effects of antiTNF agents apparently are dependent on the cell type; in some studies no detection of apoptosis induction in peripheral blood T cells could be observed, while induction of apoptosis in mucosal T cells were described in vitro and in vivo. Lastly, several findings suggest binding of antiTNF antibodies to mTNF rather than binding to sTNF is critical in IBD. In order to improve the response rates of antiTNF antibody treatment, beside pharmacokinetic reasons, it is mandatory to better understand the mechanism of action of these agents and unravel the key signaling pathways involved. First clinical studies using in vivo imaging are opening the field for individualized therapeutic approaches in the treatment of IBD.

Funding

Supported by DFG-CRC1181 -Project number (C02); and a research operating grant from the International Organization for the Study of Inflammatory Bowel Diseases.Conflict-of-interest statement: Raja Atreya has served as advisor for AbbVie and Markus F Neurath has served as advisor for MSD, AbbVie, Takeda, Boehringer, Giuliani.

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