​TNF alpha & Inflammation

Delving into TNF alpha's critical role in inflammation and its therapeutic targeting.

1. TNF alpha is a crucial cytokine in inflammation and apoptosis.
2. Its structure facilitates interaction with TNF receptors, influencing immune responses.
3. Elevated TNF alpha levels are linked to various inflammatory diseases.
4. TNF alpha activates distinct pathways through TNFR1 and TNFR2, impacting cell function.
5. Its involvement in diseases like rheumatoid arthritis and psoriasis highlights its clinical significance.
6. Therapeutic targeting of TNF alpha offers potential in treating inflammatory conditions and cancer.

Tumour necrosis factor alpha (TNF alpha) is a transmembrane protein (molecular weight of TNF alpha - 26 kDa) and a member of the TNF superfamily, with its 17 kDa secreted form mediating intracellular signalling. Originally identified in 1975 as an endotoxin-induced glycoprotein in macrophages, and later determined to be ubiquitously expressed in multiple cell types (Carswell et al., 1975), TNF alpha stimulates several critical inflammatory and apoptotic pathways by binding to one of its two membrane bound receptors, TNFR1 and TNFR2 (Aggarwall, 2003). TNF alpha levels are not detectable under healthy conditions in humans, however elevated levels of TNF alpha in tissues and serum are found in inflammatory conditions (Robak et al, 1998). TNF alpha was first cloned in 1984, and in the following two decades it has become the focus of both in vivo and in vitro experimentation, with a critical focus on its role in inducing tumourigenesis (Burugu et al, 2017; Wang et al, 2016; Pennica et al, 2005).

Tumor Necrosis Factor alpha (TNF-alpha) is a vital cytokine with diverse functions in the immune system. Primarily produced by immune cells, it plays a key role in regulating inflammation and immune responses. When infections, injuries, or other harmful stimuli are detected, TNF-alpha is released, initiating the inflammatory cascade by recruiting immune cells to the affected site. It also aids in the activation of macrophages and neutrophils to combat invading pathogens. Moreover, TNF-alpha can induce apoptosis (programmed cell death) in specific cells, contributing to the elimination of infected or damaged cells. However, dysregulated TNF-alpha production can lead to chronic inflammation and autoimmune diseases. Targeted therapies, such as TNF inhibitors, have been developed to manage inflammatory conditions by controlling TNF-alpha activity effectively.

Ligand binding to TNFR1 induces apoptosis via the caspase cascade. Specifically, the cytoplasmic death domain of TNFR1 recruits TNFR-associated death domain protein (TRADD) which forms a complex with Fas-associated death domain (FADD) and caspase 8, preceding either extrinsic or intrinsic apoptosis (mitochondrial-mediated). Extrinsic apoptosis involves the cleavage and subsequent activation of caspase 3 by active caspase 8. Intrinsic apoptosis is facilitated via the truncated form of the pro-apoptotic Bcl-2 protein Bid, leading to the translocation of Bax and Bak to the mitochondrial membrane which induces Cytochrome-c release and the formation of the apoptosome. Both extrinsic and intrinsic apoptotic pathways culminate in caspase-3 activation (Kantari and Walczak, 2011; Wang et al, 1996). As TNFR2 is death domain free, it does not induce apoptosis alone, however there is some evidence for crosstalk of both TNFR1 and TNFR2 (Naudé et al, 2007).

Ligation of TNFR1 and -2 also induce inflammatory pathway activation. TRADD association with TRAF2 and Receptor-interacting protein (RIP1) induce downstream IKK-complex activation, IKappa Beta alpha phosphorylation and degradation and the release of NF-Kappa Beta subunits inducing nuclear translocation and NF-Kappa Beta activation. Similarly, TRADD mediated recruitment of TRAF2 can activate MAPK pathways, including c-Jun N-terminal kinase (JNK) and p38, which result in activation of the transcription factor AP-1 (Blonska et al, 2005; Devin et al 2001). Additionally, there is crosstalk between inflammatory and apoptotic pathways, evident in TNF alpha-induced NF-Kappa Beta activation, which can regulate apoptosis by upregulating cFLIP levels, which in turn inhibit caspase 8 activation (Micheau et al, 2001).

Importantly, acute inflammation is a therapeutic and preventative natural defense; however, chronic inflammation is detrimental and drives a plethora of pathological conditions. TNF alpha has been identified as the critical mediator of the endotoxin-induced response, specifically facilitating that of Escherichia coli and tnfr1^-/-mice are resistant to lipopolysaccharides and Staphylococcus aureus (Pfeffer et al, 1993; Beutler et al, 1985). The inflammatory effects of TNF alpha are primarily mediated via NF-Kappa Beta. Chronic inflammation is now classified as a precursor and leading risk factor to tumorigenesis, with inflammatory signalling in the tumour microenvironment promoting tumour progression and resistance (Balkwill et al, 2005; Karin et al, 2005). Moreover, TNF alpha has been identified to play a crucial role in tumour initiation and development (Aggarwal et al, 2002; Sungamuma et al, 1999; Deigel et al, 1989). One of the mechanisms employed by TNF alpha which promotes tumour development is the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which in turn lead to DNA damage (Woo et al, 2000). Additionally, TNF alpha induces angiogenesis of tumour cells by upregulating several angiogenic factors such as vascular endothelial growth factor (VEGF) (Jing et al, 2011). Furthermore, TNF alpha promotes motility of epithelial tumour cells (Rosen et al, 1991). However, the role of TNF alpha in carcinogenesis is complex due to the range of signalling responses described above, which effectively lead to activation of 4 distinct pathways: a death domain induced pro-apoptotic pathway; an anti-apoptotic pathway, facilitated by TRAF2 associations with the E3 ubiquitin ligase cellular inhibitor of apoptosis-1 (cIAP1); MAPK-mediated AP-1 activation, and RIP1 induced NF-Kappa Beta (Chen and Goeddel, 2002). The current consensus eludes to the TNF alpha tumour microenvironment concentration and cell-specific site of elevated expression (Ohri et al, 2010).

Table 1. Overview of the involvement of TNF alpha in various inflammatory conditions.

TNF alpha offers a prime therapeutic target for cancer. To date, several drugs have been developed which target and inhibit TNF alpha, including inhibitors of TNF alpha expression (Majumdar et al, 2002); TNF alpha antibodies which bind and neutralize TNF alpha, for example infliximab, although several of these have been shown to have adverse effects (Keystone et al, 2004); and inhibitors of TNF alpha signalling primarily targeting NF-Kappa Beta (Aggarwal et al, 2006). Several methods of tumour modulation targeting TNF alpha, among other cytokines, include adoptive T cell therapy, where patients’ dendritic cells are stimulated with TNF alpha in order to induce antigen presentation (Yee et al, 2002). A similar principal has been applied for the development of dendritic cell targeted cancer vaccines which have proven to have some success in clinical trials (Ridgway, 2003). Overall, targeting cytokines, particularly TNF alpha which has the opposing roles of apoptosis and cell survival signalling, is a field of huge interest and potential with relation to cancer therapeutics and treatments for inflammatory disorders.

Figure 1: TNFa singalling pathways. TNFa binds to the membrane-bound receptors TNFR1 or TNFR2, leading to downstream apoptosis and inflammatory signalling. Activation of TNFR1 induces the formation of a death inducing signalling complex, containing TRADD, FADD and caspase-8. Formation of this complex leads to the induction of apoptosis by either the intrinsic or extrinsic pathways, both culminating in the cleavage of caspase-3. Pro-inflammatory singalling pathways can also be induced by either TNFR1 or TNFR2 activation, which signal through the adaptor protein TRAF2 to activate RIP1, NIK or MEKs, resulting in the activation of MAPK and NF-kB activation.