TNF Signaling

Question 1

The first TNFSF proteins discovered were ____ and ____.

Answer 1

TNF-α and Lymphotoxin.

Both were discovered in the 1800’s as materials present in the inflammatory mileu that caused tumor necrosis. These were the founding members of the TNF Superfamily.

Lymphotoxin is now also known as TNF-β and LT-α (as another chain it associates with, LT-β, was later discovered).

Question 2

LTα₁β₂

Answer 2

LTα may associate with LTβ to form LTα₁β₂.

This heterocomplex binds the LTβR.

Question 3

Mice lacking LTα, LTβ, or LTβR fail to develop _____.

Answer 3

Lymph nodes

Question 4

Expression of the TNFSF ligands is restricted primarily to _____, with a few notable exceptions (FasL, TL1A).

Answer 4

Immune cells (dendritic cells, activated lymphocytes, myeloid cells, etc.)

Question 5

TNFSF Ligands

Answer 5

TNFSF ligands are type II transmembrane proteins which share a TNF homology domain that facilitates trimer formation and receptor binding.

Question 6

Expression of TNFRSF receptors

Answer 6

Not restricted to immune cells, but rather appear on numerous cell types including epithelial and endothelial cells.

Question 7

TNFRSF Receptors

Answer 7

Superfamily of receptors for TNF family ligands.

The ligand-binding region is cysteine rich, typically containing 6 cysteins which form 3 disulfide bondes. Most are type I transmembrane glycoproteins with an extracellular N-terminus.

The family is subdivided into receptors that contain an intracellular death domain and receptors that contain an intracellular domain which directly recruits TRAFs (TNF receptor associated-factors).

Question 8

TNFRSF Receptor Signaling

Answer 8

Receptors are activated by aggregation (trimerization), and TNFSF ligands activate TNFRSF receptors simply by binding the heads and bringing the intracellular domains into close proximity. Hence, receptor overexpression may induce self-association and ligand-independent signaling.

Binding affinities for ligands range from the high picomolar to low nanomolar.

Question 9

Regulation of FasR/FasL Signaling

Answer 9

FasL binds to FasR (also known as CD95), which contains a death domain.

In addition to transcriptional regulation of these proteins, there exists both a decoy ligand (soluble FasL, which does not activate FasR) and decoy receptors (DCR1, DCR2, DCR3, and OPG).

Question 10

HVEM

Answer 10

Herpesvirus entry mediator, also known as TNFRSF14.

Involved in two distinct entry pathways for two distinct Herpesviruses, Herpes simplex virus and cytomegalovirus.

Physiologically, HVEM can bind an array of different TNFSF ligands to different effects.

Question 11

HVEM Signaling

Answer 11

HVEM displays complex signaling behavior, as it can both promote and inhibit apoptosis. There are two discrete faces of the receptor.

LIGHT and LTα bind one face to trigger signaling that promotes survival and differentiation. BTLA may bind the other face to restrict proliferation and survival by recruitment of tyrosine phosphatase SHP1/2.

If both ligands are bound simultaneously, SHP1/2 is not recruited and the survival signaling wins out.

Question 12

TNFR1 Signaling

Answer 12

When TNF-α binds TNFR1, the death domain is activated. Upon activation, the DD recruits TRADD and FADD proteins, which in turn recruit and acivate caspase 8. Caspase 8 proceeds to activate the executioner caspases 3, 6, and 7, which initiate the classical apoptosis pathway.

Fas signaling operates by the same pathway.

Question 13

TNFR2

Answer 13

TNFR2 does not contain a death domain, but instead initiates pro-survival signaling through the PI3K-Akt-NFkB pathway. In regulatory T cells, TNFR2 binding also activates STAT5 and triggers TGF-β and IL-10 transcription.

Survival of a given cell expressing both TNFR1 and TNFR2 depends on the ratio of the two receptors and on the concentration of TNF-α, with low [TNF-α] favoring survival and high [TNF-α] favoring apoptosis.

Question 14

LIGHT

Answer 14

TNFSF ligand.

Upregulates cell adhesion molecules, IL-8, and COX-2 in epithelial cells through LTβR and HVEM. Helps mediate transition out of monocytic inflammation by inducing MΦ apoptosis and expressio of TGF-β and IL-13, encouraging fibrosis and tissue remodeling.

Question 15

RANKL

Answer 15

TNFSF member.

Induces cell adhesion molecule expression and ROS/RNS production in endothelial cells. This both activates endothelial cells and promotes angiogenesis.

Required for lymph node development, similar to LT signaling proteins.

Question 16

TRAIL

Answer 16

TNFSF member.

Activates RNS in epithelial cells and induces prostanoid synthesis, as well as epithelial cell proliferation. Has no effect on cell adhesion molecules and does not signal through NFkB.

Question 17

Regulation of Neutrophils by TNF Signaling

Answer 17

Neutrophils express the death-domain containing TNFRSF members TNFR1, FasR, and TRAIL-R2, but also express the anti-apoptotic TNFR2. Thus, they respond to both pro- and anti-apoptotic signals from their environmet.

In the presence of TNFα, endothelial cells (which express FasL at baseline) will down-regulate FasL and up-regulate cell adhesion molecules to recruit neutrophils. Upon extravasation, neutrophils down-regulate TNFR1, but maintain Fas expression. This facilitates neutrophil survival while inside an acutely inflamed tissue.

However, once FasL-expressing MO and MΦ arrive, Fas-mediated apoptosis begins to aid in efferocytosis. TRAIL is expressed at this time, inducing neutrophil apoptosis without activating NFkB or cell adhesion, making it important to the neutrophilic to monocytic inflammation transition.

OX40L-signaling may compete with Fas and TRAIL signaling to prolong the inflammation phase.

Question 18

TNFRSF members __________ are required for the development of T regs in the thymus.

Answer 18

TNFR2, GITR, and OX40.

In mature T regs, TNFR2 signaling induces proliferation and enhances suppressive ability.

Question 19

BAFF and APRIL

Answer 19

Required factors for B cell survival, maturation, and (with CD40L and OX40L) isotype class switching.

CD40L is particularly important for differentiation into plasma cells.

Question 20

IBD Risk Alleles from the TNFSF and TNFRSF.

Answer 20

FasL, GITR-L, TL1A, HVEM, GITR, OX40, 4-1BB/CD137, CD40, and DCR3.

Question 21

DCR3

Answer 21

A “decoy receptor” for FasL, TL1A, and LIGHT.

Expressed at high levels in the inflamed mucosa and serum of Crohn’s and ulcerative colitis patients. Upregulation of DCR3 is considered a self-protective, anti-inflammatory mechanism.

Question 22

Clinically Approved TNF inhibitors

Answer 22

Etanercept, Infliximab, Adalimumab, Golimumab, and

Certolizumab.

All protein-based, and such are prone to the development of antibodies, resulting in a decrease in efficacy over time in some patients. The principal mechanism of action for all drugs is sequestration of TNF-α, although IgG1-Fc-mediated cytotoxicity pathways may also contribute to efficacy. Show a clinical response in ~70% of arthritis and IBD patients.

Ineffective in treating systemic lupus erythematosus, Sjorgen’s syndrome, and osteoarthritis. Can exacerbate symptoms of multiple sclerosis.

Question 23

Adalimumab and Golimumab

Answer 23

Human anti-TNF-α IgG1 antibodies.

Question 24

Etanercept

Answer 24

Fusion protein of TNFR2’s TNF-α-binding domain and the IgG1 Fc region. Blocks LTα as well as TNF-α, as opposed to all other approved drugs which are TNF-α-specific antibodies.

Question 25

Inflixumab and Certolizumab

Answer 25

Humanized mouse anti-TNF-α.

Contains the human IgG1 Fc domain.