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Tumor necrosis factor ( TNF), formerly known as TNF-α, is a chemical messenger produced by the immune system that induces inflammation. TNF is produced primarily by activated macrophages, and induces inflammation by binding to its receptors on other cells. It is a member of the tumor necrosis factor superfamily, a family of transmembrane proteins that are Cytokine, chemical messengers of the immune system. Excessive production of TNF plays a critical role in several inflammatory diseases, and TNF inhibitors are often employed to treat these diseases.
TNF is produced primarily by macrophages but is also produced in several other cell types, such as T cells, B cells, dendritic cells, and mast cells. It is produced rapidly in response to pathogens, cytokines, and environmental stressors. TNF is initially produced as a type II transmembrane protein (tmTNF), which is then cleaved by TNF alpha converting enzyme (TACE) into a soluble form (sTNF) and secreted from the cell. Three TNF molecules assemble together to form an active homotrimer, whereas individual TNF molecules are inert.
When TNF binds to its receptors, tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2), a pathway of signals is triggered within the target cell, resulting in an inflammatory response. sTNF can only activate TNFR1, whereas tmTNF can activate both TNFR1 and TNFR2, as well as trigger inflammatory signaling pathways within its own cell. TNF's effects on the immune system include the activation of white blood cells, blood coagulation, secretion of cytokines, and fever. TNF also contributes to homeostasis in the central nervous system.
Inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease can be effectively treated by drugs that inhibit TNF from binding to its receptors. TNF is also implicated in the pathology of other diseases including cancer, liver fibrosis, and Alzheimer's, although TNF inhibition has yet to show definitive benefits.
In the 1980s, TNF was purified, sequenced, and cloned in bacteria. Studies on recombinant TNF confirmed the anticancer potential of TNF, but this optimism faded when TNF injections were found to induce endotoxin shock. TNF was also discovered to be the same protein as cachectin, known to cause muscle wasting in mice. These findings demonstrated that TNF could be detrimental in excessive quantities. In 1992, TNF antibodies were found to reduce joint inflammation in mice, revealing TNF's role in inflammatory diseases. This led to the approval of the first anti-TNF therapy for rheumatoid arthritis in 1998.
The TNF gene is very similar among mammals, ranging from 233 to 235 amino acids. The TNF proximal promoter region is also highly conserved among mammals, and nearly identical among higher primates. The similarity of the TNF gene among fish is lower, ranging from 226 to 256 amino acids. Like mammalian TNF, the fish TNF gene has been shown to be stimulated in macrophages by antigens. All TNF genes have a highly conserved C-terminal module known as the TNF homology domain, due to its important role in binding TNF to its receptors.
TNF gene expression is regulated by a proximal promoter region consisting of approximately 200 base pairs. Most of the binding sites within the proximal promoter region can recognize multiple transcription factors, enabling TNF to be activated by a variety of signaling pathways. As transcription factors bind to the promoter region, they also bind to coactivators, assembling into a large structure known as an enhanceosome. The composition of the enhanceosome depends on ambient factors within the cell, particularly nuclear factor of activated T-cells (NFAT).
TNF expression is also regulated by DNA structure. DNA is coiled around histones, which is loosened by acetylation and condensed by methylation. Proteins that acetylate histones at the TNF promoter, particularly CREB-binding protein in T cells, are often critical for TNF expression. In contrast, several cell types that do not express TNF are highly methylated at the histones of the TNF promoter. Long-range intrachromosomal interactions can also regulate TNF expression. In activated T-cells, the DNA surrounding the TNF promoter circularizes, bringing promoter complexes closer together and enhancing transcription efficiency.
The secreted extracellular portion, denoted sTNF, consists of 157 amino acids. Unlike tmTNF, sTNF can only bind to TNFR1. The secondary structure of sTNF consists primarily of alternating strands that join into two sheets, known as antiparallel β-sheets. The two sheets are layered on top of each other, forming a wedge shape known as an antiparallel β-sandwich. Remarkably, this structure is similar to those seen on the coats of viruses. The last 9 residues of the C-terminus are locked into the middle strand of the bottom sheet, and are necessary for bioactivity.
Both tmTNF and sTNF are only bioactive as homotrimer, whereas individual monomers are inactive. The rate at which TNF trimers disassemble is constant, whereas the rate at which TNF trimers assemble increases with TNF concentration. This causes TNF to be mostly trimers at high concentrations, whereas TNF is mostly monomers and dimers at low concentrations. The coexistence of TNF dimers and trimers in dynamic equilibrium suggests that TNF might be a morpheein. Small molecules that stabilize TNF dimers and prevent the assembly of TNF trimers present a potential mechanism for inhibiting TNF.
Upon activation by TNF, TNFR1 trimerizes and forms complex I by recruiting RIPK1 and TRADD, which recruits TRAF2, cIAP1 and cIAP2, and LUBAC. cIAP1 and cIAP2 are ubiquitin ligases that form K63-linked ubiquitin chains, which recruit TAK1 via TAB2 and TAB3. LUBAC is also a ubiquitin ligase that forms M1-linked ubiquitin chains, which attract IKK via IKBKG. TAK1 activates the MAPK pathways, as well as IKK, which in turn activates the canonical NF-κB pathway. The MAPK pathways and the NF-κB pathway activate multiple transcription factors in the nucleus, which result in cell survival, proliferation, and inflammatory response. Complex I is negatively regulated by deubiquitinases such as A20, CYLD, and OTULIN, which destabilize complex I.
Complex II is formed when RIPK1 and/or TRADD disassociate from complex I and bind with FADD to activate caspase 8, leading to cell death. Complex IIa includes TRADD and can activate caspase 8 without RIPK1, while complex IIb does not include TRADD, so it is dependent on RIPK1 for the activation of caspase 8. The pathways of complex I induce three checkpoints that prevent complex II from inducing cell death.
In the first checkpoint, IKK disables RIPK1 via phosphorylation while it is attached to complex I. This disables complex IIb, which is dependent on RIPK1. Since IKK is dependent on the ubiquitination of complex I, conditions that affect ubiquitination, such as inhibition of cIAP1/2 and LUBAC, mutation of the RIPK1 ubiquitin acceptor site, or deficiencies of A20 and OUTLIN, can disable this checkpoint. The disabling of the IKK checkpoint activates complex IIb, leading to apoptosis, or pyroptosis by cleaving GSDMD. The disabling of the IKK checkpoint can also indirectly activate complex IIa by disabling the NF-κB pathway, which controls the second checkpoint.
In the second checkpoint, the NF-κB pathway promotes the expression of pro-survival genes such as CFLAR, which counteracts the activation of caspase 8 in complex IIa. This checkpoint can be disabled by translation inhibitors such as cycloheximide, as well as by the disabling of the IKK complex, which controls the NF-κB pathway. The disabling of this checkpoint activates complex IIa, leading to apoptosis.
In the third checkpoint, non-lethal caspase 8 is activated by TNFR1 signalling, which binds to complex IIb and cleaves RIPK1, disabling it. It is unknown why this form of caspase 8 does not cause cell death. The disabling of this checkpoint, via inactivation of caspase 8, causes RIPK1 from complex IIb to bind to RIPK3 and MLKL, forming complex IIc, also referred to as the necrosome. The necrosome then causes necroptosis.
Upon binding to tmTNF, TNFR2 trimerizes and directly recruits TRAF2, as well as TRAF1 or TRAF3. TRAF2 is central to the TNFR2 signaling complex and recruits cIAP1/2. If there is an accumulation of NIK within the cell, TRAF2/3 and cIAP1/2 may be formed as a complex with inactive NIK. When TRAF2/3 binds to TNFR2, the attached NIK is activated, which in turn activates CHUK. This allows p100 and RelB to be processed into a heterodimer which activates the non-canonical NF-κB pathway, leading to cell proliferation. The expression of p100 and RelB is potentiated by the activation of the canonical NF-κB pathway by TNFR1. Thus, TNFR2 non-canonical NF-κB activation is dependent on the canonical NF-κB activation by TNFR1, as well as the accumulation of NIK within the cell.
TNFR2 can also activate the canonical NF-κB pathway, though this is less common than non-canonical NF-κB activation. The details of TNFR2's activation of the canonical NF-κB pathway are unknown. Presumably, TAK1 and IKK are recruited by the TRAF2 / TRAF1/3 / cIAP1/2 signalling complex, which in turn activates the canonical NF-κB pathway.
TNFR2 can indirectly induce cell death by degrading cIAP1/2 as part of the non-canonical NF-κB pathway. The degradation of cIAP1/2 affects the ubiquitination of the TNFR1 signalling complex, which inhibits the function of IKK. This disables the IKK cell death checkpoint in TNFR1, inducing cell death.
In tumor cells, such as B-cell lymphoma, tmTNF reverse signalling has been shown to increase NF-κB activity, enhancing cell survival and apoptosis resistance. In natural killer cells, tmTNF reverse signalling increases cytotoxic activity by increasing the expression of perforin, granzyme B, Fas ligand, and TNF. In T cells, the activation of the JNK pathway by tmTNF reverse signalling can lead to cell cycle inhibition and apoptosis.
In monocytes, tmTNF has been shown to play a dual role in mediating the monocyte's inflammatory response to sTNF. If tmTNF reverse signalling occurs before a monocyte is activated by sTNF, then the monocyte's inflammatory response to sTNF is enhanced. If tmTNF reverse signalling occurs after a monocyte is activated by sTNF, then the inflammatory response is reduced. Meanwhile, tmTNF reverse signalling reduces a monocyte's inflammatory response to endotoxin. This effect is caused by tmTNF activating the JNK and p38 pathways, which induces TGF-β production, which then interferes with the signalling pathway of endotoxin.
TNF is the principal cytokine for regulating acute inflammation, though many of its functions are shared with other cytokines, especially IL-1. By binding to TNF receptors, TNF can perform functions including stimulating endothelial cells to induce coagulation, which obstructs blood flow to prevent the spread of microbes; stimulating endothelial cells and macrophages to secrete chemokines that attract white blood cells; stimulating the secretion of other cytokines such as IL-1; activating neutrophils and macrophages; stimulating the liver to produce acute phase proteins, such as C-reactive protein; inducing catabolism of muscles and fat to produce energy; and stimulating scar tissue formation, also known as fibrosis. In addition to inducing the secretion of cytokines, TNF itself can be induced by cytokines, enabling a cascade of inflammatory signals. Excessive amounts of TNF can cause septic shock.
Much of TNF's functions are mediated through inflammatory signalling pathways, such as MAPK and NF-κB. Many pathogens attempt to prevent an immune response by hijacking cells and disrupting their inflammatory pathways. In response to this, the TNFR1 signalling pathway has cell death pathways that are inhibited by the activities of the inflammatory pathways. If a cell's inflammatory pathways are disrupted, the cell death pathways are uninhibited, triggering cell death. This prevents the pathogen from replicating within the cell, as well as alerting the immune system.
Additionally, TNF induces fever to help the body fight infections. TNF can induce fever by triggering the release of cytokines interleukin-1 and interleukin-6, or through other mediators like PLA2. TNF or its mediators can reach the hypothalamus either through circulation in the bloodstream or through secretion by macrophages and endothelial cells near the hypothalamus. TNF can also induce fever by stimulating the Vagus nerve in the liver, which signals to neurons to secrete norepinephrine. All of these pathways culminate in the synthesis of prostaglandins, which interact with the OVLT in the hypothalamus to raise the target temperature of the body.
Through TNFR1 signalling, TNF can increase the surface expression of and NMDA receptor in neurons, strengthening synaptic transmission. TNF also decreases the surface expression of , reducing the activity of inhibitory synapses. TNF can also modulate the release of glutamate, an excitatory neurotransmitter, and S100B, a zinc-binding protein, by astrocytes. The modulation of excitation and inhibition of neurons by TNF indicates that TNF plays a role in synaptic scaling and plasticity.
Through TNFR2 signalling, TNF promotes the proliferation and maturation of oligodendrocytes, which produce protective myelin sheaths around nerve cells. On the other hand, TNF becomes cytotoxic to oligodendrocyte progenitor cells when the cells are in contact with astrocytes.
TNF induces inflammation both by activating inflammatory pathways, as well as by triggering cell death. Cell death triggers inflammation by exposing the components of dying cells to neighboring cells, as well as by compromising barrier integrity in the skin and intestine, allowing microbes to infiltrate the tissue. TNF is believed to trigger cell death in inflammatory diseases due to elevated levels of interfering cytokines, elevated levels of TNFR2 signalling, or genetic mutations. Drugs that target proteins involved in TNF-induced cell death, such as RIPK1, are being evaluated for their efficacy against autoinflammatory diseases.
In some cancers, TNF has been shown to play an inhibitory role, primarily when injected locally, repeatedly, and at high concentrations. Due to TNF's adverse side effects, potential TNF cancer treatments seek to maximize cytotoxicity to tumors while minimizing exposure to the entire body. Some treatments increase cytotoxicity by inhibiting the cell survival pathways of tumors before treatment with TNF. Other treatments localize TNF activity using antibody-TNF fusions, also known as immunocytokines. Local TNF treatment has been shown to induce tumor regression, though they rarely induce complete remission. Body-wide administration of TNF has shown low efficacy and high side effects.
In many cancers, TNF is believed to play a supportive role. High TNF expression levels are associated with more advanced cancers, and TNF expression is found in tumor cells from the early stages of disease. TNF expression can lead to the recruitment of white blood cells that promote metastasis, as well as direct activation of pathways that promote tumor survival, invasion, and metastasis. TNF-blockers such as infliximab and etanercept did not induce a response in most advanced or metastatic cancers, but some studies have shown disease stabilization.
Conversely, TNF plays a role in the progression of HIV by inducing apoptosis of T cells in HIV-infected people. TNF blockage has reportedly led to clinical improvement in HIV without worsening the infection, though data is limited.
Although TNF blockers showed efficacy in treating sepsis in mice, they showed mixed results in treating sepsis in humans. This is believed to be due to the dual role that TNF plays in the immune system; blocking TNF reduces the severe inflammation that causes sepsis, but also hinders the immune system's ability to resist the infection. It is hypothesized that TNF blockers are more beneficial in cases of severe sepsis, where the probability of death is higher.
Additionally, hepatocyte death, the initial event that drives liver injury and fibrosis, may be induced by TNF, though this connection is uncertain. TNF injection alone does not induce hepatocyte death in vivo. However, when TNF injection is coupled with survival pathway inhibition, such as during hepatitis C virus infection, TNF induces hepatocyte death and acute liver failure. The remnants of dead hepatocytes are consumed by HSCs and Kupffer cells, which then secrete fibrosis-promoting factors, such as TGF-β, as well as promoting further hepatocyte death.
Paradoxically, TNF-blockers can cause demyelination of neurons and worsen multiple sclerosis symptoms. This is believed to be due to the homeostatic role of TNF in the central nervous system, especially on neuron myelination via TNFR2. The selective blockade of TNFR1 has shown positive outcomes in animal models.
TNF-induced neuroinflammation has also been associated with Alzheimer's disease, and is suspected to contribute to the Amyloid plaques and tau protein hyperphosphorylation found in the brains of Alzheimer's patients. TNF blockers have been associated with reduced risk of developing Alzheimer's. Some studies have shown TNF blockers to slightly improve cognition in Alzheimer's patients, though larger studies are needed. Since TNF blockers cannot pass through the blood–brain barrier, it is believed that reducing TNF levels across the body also reduces TNF levels within the brain.
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