8: Tumor Necrosis Factor Alpha

Tumor Necrosis Factor Alpha (TNFα)

• Signaling protein involved in systemic inflammation
• Primary regulator of immune system cells
• Acts as a pyrogen, inducing fever and apoptotic cell death
• Cytotoxic factor
• Secreted from macrophages and other cell types
• Produced as a transmembrane protein
• Undergoes protease cleavage
• Results in a soluble, mature protein version
• Discovered by Dr. Lloyd J. Old (1933-2011) of Sloan-Kettering, William Bradley Coley (1862-1936) of UC Irvine, and Dr. Nancy H. Ruddle of Yale University

TNFα Receptors and Associated Pathways

• Binds to two receptors: TNFR1 and TNFR2
• These receptors associate with a protein complex that includes the adaptor protein TRADD
• Activation of caspase-mediated cell death
• Activation of NF-KB transcription factor
• Involved in inflammatory response and cell regulation
• Activation of MAPK pathways
• Leads to an immediate-early response to stress

TNF-α and Allergic Response

• Implicated in allergic respiratory disease
• Characterized by complex cytokine interactions, airway inflammation, reduced breathing ability, and airway remodeling in response to allergens

Mild-Moderate Asthma

• Involves Th2 lymphocytes, eosinophils, and elevated IgE levels
• Treatable with corticosteroids

Severe Refractory Asthma

• Involves Th1 and Th17 lymphocytes, neutrophils, eosinophils, and TNF-α
• Not treatable by corticosteroids
• Exhibits heterogeneous phenotypes

TNF-α and Asthma

• Affects the respiratory system by recruiting pro-inflammatory cells
• Induces airway remodeling, leading to typical asthma symptoms
• Inhaled recombinant human TNF-α reduces forced expiratory volume (FEV)

Influenza

• Self-assembling structure made of viral proteins and nucleic acids
• Surrounded by a membrane envelope
• Enveloped virion contains a helical nucleoprotein capsid

RIPK3 & IAV

• RIPK3-mediated cell death and inflammasome pathways are essential for clearing influenza A virus (IAV) infections
• IAV strongly induces TNF-α in the infected lung
• Mice lacking both FADD and MLKL are more susceptible to IAV
• TNF-α is critical for clearing influenza from infected lungs
• RIPK3-independent apoptosis, relying on FADD and caspase-8, is engaged by IAV
• Functions with RIPK3-driven cell death and inflammasome activation to eliminate the infected cell and limit virus spread

IKK Activity and Gene Expression

• A small number of mammalian signaling pathways mediate diverse physiological responses to cellular stimuli
• Stimulus-specific signaling is mediated by the IKK-NF-κB signaling module
• Temporal control of signaling by IKK, its substrate inhibitor NF-κB, and transcription factor NF-κB allows for selective gene activation
• Distinct inflammatory stimuli induce distinct IKK profiles
• TNFα-induced IKK activity is rapidly attenuated by negative feedback
• LPS signaling and gene expression depend on a cytokine-mediated positive feedback mechanism
• The functional pleiotropism of NF-kB is based on the responsiveness of IKK to diverse signals transduced by plasma membrane-bound receptors or subcellular organelles
• IKK profiles are measured by IKK IP-kinase assay in mouse embryonic fibroblasts (MEFs) stimulated with a 45-min pulse of either TNF (1 ng/ml) or LPS (0.1 mg/ml)
• Stimulus-induced increases in cytokines and chemokines are induced by a factor of greater than 10 over baseline in the first hour of LPS stimulation

LPS Signaling Control

• IKK activity is regulated by negative and positive feedback loops
• A20-mediated negative feedback loop occurs in the TNFR signaling pathway
• A positive feedback/feedforward loop in the LPS signaling pathway is controlled by NF-κB and possibly IRF3
• Results in TNFα expression
• The paper shows that TNF and possibly other cytokines mediate feed-forward mechanisms
• This occurs in response to endotoxin challenges that produce positive feedback on IKK activity
• A feed-forward mechanism is critical for stimulus-specific gene expression
• The sensitivity to the temporal profile of IKK activity suggests that the underlying molecular mechanisms may provide sensitive signaling nodes for cellular signaling cross-talk

NF-kB Single Cell Microscopy

• 3T3 mouse fibroblast cells were cultured in a microfluidic cell culture platform
• Used to measure NF-kB activity under ten different TNF-a concentrations (100 ng/ml to 0.005 ng/ml) with single cell resolution
• Upon stimulation, NF-kB was transported from the cytoplasm to the nucleus and back out again in characteristic oscillations

TNF-α Stimulation of NF-kB

• The nuclear localization dynamics of NF-kB, in response to stimulation by TNF-α, were mapped
• Single-cell traces, time-dependent gene expression profiles were measured across a concentration range of four orders of magnitude.
• Cells activate in response to TNF-α in a digital manner
• Fraction of activated cells decreases to zero with decreasing TNF-α dose

Gene Expression Conclusions

• Early gene expression does not depend on inducing signal intensity
• Later gene expression requires persistent nuclear localizations of the transcription factor at the highest input signal levels
• Cells process analogue signal intensity information using the timing, intensity, and number of oscillations
• Used to generate digital outputs (activation and early gene expression.)