Fig 1.
Cytokine Gradient around the Initial Site of Infection in Macrophages.
The cytokine gradient or field around the primary infected cell (red) coordinates with the mechanism of the proinflammatory response. As the initially infected macrophage secretes TNF-α (yellow), the TNF-α gradient is highest around the initial cell and radiates outward whereas the IFN-γ (blue) field, which is primarily secreted by peripheral NK cells radiates towards the site of infection. These cytokine fields result in three pre-stimulatory conditions from which cells exhibit dynamically unique proinflammatory responses upon LPS stimulation [30, 35].
Fig 2.
Reaction Scheme of LPS Mediated Membrane Complex Activation.
Six reversible reactions capture the mechanism of LPS binding and activation of macrophages. The numbers in parenthesis denote the number of individual reactions implemented in the model to represent a specific binding/unbinding reaction. In short, LPS can activate macrophages in two ways, either TLR4 mediated or TLR4 and MD2 mediated, the latter of which is significantly faster and more robust.
Fig 3.
LPS Activated Cytokine Synergy on iNOS gene Expression and NO Production.
LPS stimulation activates transcription factors AP1 and NF-κB, which in turn activates the TNF-α gene. Translated TNF-α exports to the extracellular compartment and activates the TNF-α-receptor 1 that proceeds to further stimulate AP1 and NF-κB via an autocrine loop. NF-κB can also activate IRF1, which increases production of TNF-α, however, the production of IRF1 is greatly increased by IFN-γ stimulation and STAT1 phosphorylated dimer activation (bold arrows). Therefore, the synergistic expression of TNF-α and IRF1 by IFN-γ greatly amplifies iNOS gene expression and NO production.
Fig 4.
iNOS Gene Expression Mechanism.
The Enzyme term in this figure refers to the DNA-RNA Polymerase complex. Dissociation constants are labeled accordingly and their values can be found in the S1 Table.
Table 1.
Significant Parameters that Drive the System.
Fig 5.
TNF-α Stimulated iNOS mRNA and Transcription Factor Expression Dynamics.
The system was simulated with LPS (control), TNF-α, and TNF-α with LPS as inputs. (A-D) show the transcription factors of iNOS under the three stimulatory conditions. STAT1 phosphorylated dimers remain at zero since there is no IFN-γ stimulation. (E) Shows the expression of iNOS under LPS stimulation only. The delay in iNOS expression correlates to LPS causing a delayed activation of iNOS in the absence of other cytokines due to a larger activation cascade. (F) iNOS mRNA gene expression under TNF-α and TNF-α/LPS stimulation respectively.
Fig 6.
IFN-γ Stimulated iNOS mRNA and Transcription Factor Expression Dynamics.
The system was stimulated with IFN-γ, IFN-γ /TNF-α, IFN-γ and LPS, and IFN-γ /TNF-α and LPS as inputs. (A-D) show the transcription factors of iNOS under the four stimulatory conditions. STAT1 phosphorylated dimers are expressed during IFN-γ stimulation (D) and the dynamics are similar to previous models [16]. (E) Shows the expression of TNF-α mRNA under IFN-γ stimulation. IFN-γ by itself is able to activate TNF-α gene expression through IRF1. (F) iNOS mRNA gene expression under the four stimulatory conditions. The magnitude of expression is much greater under IFN-γ stimulation as opposed to TNF-α only activation (Fig 4F), which may be attributed to the activation and compounding effect of TNF-α gene expression in the presence of IFN-γ.
Fig 7.
Maximum iNOS mRNAn values were obtained through model simulation under various conditions. Expression levels are relative to LPS control stimulation. The greatest increase in maximum iNOS production can been seen between TNF-α stimulation and IFN-γ stimulation. However, in the presence of LPS, there is only a slight increase in iNOS gene expression when compared to cytokine activation levels.
Fig 8.
Time to Peak iNOS mRNA Expression.
The model was simulated under the seven activation conditions and the time to peak iNOS gene expression was obtained for each simulation. The bar graph suggests that LPS is the slowest activator of iNOS whereas TNF-α is the fastest. This feature highlights the need for autocrine regulation of TNF-α during the proinflammatory response to help fight intracellular pathogens. IFN-γ however shows a delayed response when compared to TNF-α. This delayed response may be beneficial to boost the proinflammatory response following TNF-α stimulation.
Table 2.
Peak and Average iNOS and NO Expression Levels.
Fig 9.
TNF-α and IFN-γ primed iNOS Gene Expression Dynamics.
The model was simulated with TNF-α, IFN-γ, or both TNF-α /IFN-γ for 24 simulation hours to prime the system. The concentrations of iNOS transcription factors, iNOS mRNA, and iNOS protein were used as inputs to the system for an 8-hour addition simulation under LPS activation. The resulting dynamics display the behavior of iNOS gene expression under primed conditions. The combination of both, TNF-α and IFN-γ causes a significant increase in the dynamics of the primed system however, IFN-γ priming has the ability to generate an increase in the proinflammatory burst when compared to the initial value of iNOS gene expression after IFN-γ priming.
Fig 10.
iNOS Gene Expression as a Function of TNF-α or IFN-γ.
A range of concentrations of TNF-α and IFN-γ were used to stimulate the system and the maximum iNOS gene expression rates were plots as a function of cytokine concentration. The time to reach maximal expression under varying concentrations of cytokines was used to derive the velocity of expression under each respective condition. (A) The Michaelis-Menten type plot shows the rate of iNOS gene expression as a function of TNF-α. Since TNF-α induces peak iNOS expression faster than IFN-γ, the rate of expression is greater for TNF-α than for IFN-γ. (B) The Michaelis-Menten type plot shows the rate of iNOS gene expression as a function of IFN-γ. The Km value is marked at half-maximal velocity. (C) The expanded graph of TNF-α dependent iNOS gene expression rate shows a sigmoidal curve suggesting a cytokine threshold for gene activation. (D) The expanded graph of IFN-γ dependent iNOS gene expression rate also shows a sigmoidal curve in addition to a smaller range upon which Vmax is reached. Vmax values are in arbitrary units, which correlate to the change of relative expression per time.
Fig 11.
The consolidated signal transduction system represented here shows how IFN-γ priming directly stimulates the system to ultimately generate iNOS.