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Cited by 210Immune Response and Inflammationinterferon and immune responsesGene Regulatory Network Analysis

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Investigating the Role of TNF-α and IFN-γ Activation on the Dynamics of iNOS Gene Expression in LPS Stimulated Macrophages

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Salim, Taha, Sershen, Cheryl L., May, Elebeoba E.·2016·PLOS ONE
DOI 10.1371/journal.pone.0153289PMC PMC4898755
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Abstract

Macrophage produced inducible nitric oxide synthase (iNOS) is known to play a critical role in the proinflammatory response against intracellular pathogens by promoting the generation of bactericidal reactive nitrogen species. Robust and timely production of nitric oxide (NO) by iNOS and analogous production of reactive oxygen species are critical components of an effective immune response. In addition to pathogen associated lipopolysaccharides (LPS), iNOS gene expression is dependent on numerous proinflammatory cytokines in the cellular microenvironment of the macrophage, two of which include interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To understand the synergistic effect of IFN-γ and TNF-α activation, and LPS stimulation on iNOS expression dynamics and NO production, we developed a systems biology based mathematical model. Using our model, we investigated the impact of pre-infection cytokine exposure, or priming, on the system. We explored the essentiality of IFN-γ priming to the robustness of initial proinflammatory response with respect to the ability of macrophages to produce reactive species needed for pathogen clearance. Results from our theoretical studies indicated that IFN-γ and subsequent activation of IRF1 are essential in consequential production of iNOS upon LPS stimulation. We showed that IFN-γ priming at low concentrations greatly increases the effector response of macrophages against intracellular pathogens. Ultimately the model demonstrated that although TNF-α contributed towards a more rapid response time, measured as time to reach maximum iNOS production, IFN-γ stimulation was significantly more significant in terms of the maximum expression of iNOS and the concentration of NO produced.

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Figures

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 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].CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
Fig 7 Maximum iNOS Expression. 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 7 Maximum iNOS Expression. 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
Fig 11 Mechanism of IFN-γ Priming. The consolidated signal transduction system represented here shows how IFN-γ priming directly stimulates the system to ultimately generate iNOS.
Fig 11 Mechanism of IFN-γ Priming. The consolidated signal transduction system represented here shows how IFN-γ priming directly stimulates the system to ultimately generate iNOS.CC BY · PubMed Central

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Metadata

Authors
Salim, T., Sershen, C. L., & May, E. E.
Affiliations
University of Houston (US)
Corresponding author
May, Elebeoba E.
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2016-06-08
Volume
11
Issue
6
Pages
e0153289
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0153289
PMC
PMC4898755
OpenAlex
W2415587673
Cited by
210
References
94
Citations by year
2026: 22 · 2025: 24 · 2024: 29 · 2023: 22 · 2022: 26 · 2021: 31 · 2020: 26 · 2019: 14 · 2018: 10 · 2017: 5 · 2016: 1
FWCI
6.05
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
  • Defense Threat Reduction Agency (FA8650-10-2-6062 subaward #2381 (Henry M. Jackson Foundation))
  • Sandia National Laboratories, National Nuclear Security Administration (LDRD 151348)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
Keywords
inducible nitric oxide synthase, iNOS expression, macrophages, lipopolysaccharide stimulation, interferon-gamma, TNF-α, nitric oxide production, IRF1, cytokine priming, intracellular pathogens, reactive nitrogen species, systems biology models, pro-inflammatory response
MeSH terms
Humans, Interferon-gamma, Lipopolysaccharides, Macrophages, Nitric Oxide, Tumor Necrosis Factor-alpha, Gene Expression Regulation, Enzymologic, Models, Immunological, Nitric Oxide Synthase Type II

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