A Method for Deciphering Major Drivers of Bacterial Iron Stress Response in the Presence of Oxidative Stressors.

This paper describes a method for deciphering major drivers of bacterial stress response using an empirically informed computational approach. We develop a working model of iron flux regulation and concomitant oxidative stress response in Escherichia coli. The integrated model is used to investigate the temporal effects of iron and hydrogen peroxide stress on bacterial growth and metabolism. We employ a sensitivity analysis platform and, using various measures, probe for major mechanistic drivers of the bacterial response to iron stress.

Compounding effect of vitamin D3 diet, supplementation, and alcohol exposure on macrophage response to mycobacterium infection.

Vitamin D3 is known to be a key component in the defense against Mycobacterium tuberculosis (Mtb) infection through the regulation of cytokine and effector molecules. Conversely, alcohol exposure has been recognized as an immune dysregulator. Macrophages were extracted from D3 deficient and sufficient diet mice and supplemented with D3 or exposed to ethanol during ex vivo infection using M. bovis BCG, as a surrogate for Mtb. Results of our study indicate that while exogenous supplementation or alcohol exposure did alter immune response, in vivo diet was the greatest determinant of cytokine and effector molecule production. Alcohol exposure was found to profoundly dysregulate primary murine macrophages, with ethanol-exposed cells generally characterized as hyper- or hyporesponsive. Exogenous D3 supplementation had a normative effect for diet deficient host, however supplementation was not sufficient to compensate for the effects of diet deficiency. Vitamin D3 sufficient diet resulted in reduced cell cytotoxicity for the majority of time points. Results provide insight into the ramifications of both the individual and combined health risks of D3 deficiency or alcohol exposure. Given the clinical relevance of D3 deficiency and alcohol use comorbidities, outcomes of this study have implications in therapeutic approaches for the treatment of tuberculosis disease.

Spatiotemporal Analysis of Mycobacterium-Dependent Macrophage Response.

There are three main outcomes of Mycobacterium tuberculosis infection: clearance, dissemination, and containment - in which the immune system physically isolates the invading microbes in lesions called granulomas. These structures are a hallmark of the disease and play an important role in the progression of infection. However, current in vitro and in vivo methods are ill adapted for spatial and temporal quantification of host-pathogen dynamics, which are necessary for the development of granulomas. We have developed an integrated 3D in vitro and computational platform with longterm time-lapse confocal imaging to provide a semi-automatic analysis of host-pathogen interaction data. Through exploratory data analysis, we conduct a preliminary investigation of how the intracellular bacterial load of macrophages can impact cellular spatiotemporal dynamics during Mycobacterium infection.

The dynamic immunomodulatory effects of vitamin D3 during Mycobacterium infection.

Mycobacterium tuberculosis ( Mtb), is a highly infectious airborne bacterium. Previous studies have found vitamin D3 to be a key factor in the defense against Mtb infection, through its regulation of the production of immune-related cytokines, chemokines and effector molecules. Mycobacterium smegmatis was used in our study as a surrogate of Mtb. We hypothesized that the continuous presence of vitamin D3, as well as the level of severity of infection would differentially modulate host cell immune response in comparison with control and the vehicle, ethanol. We found that vitamin D3 conditioning promotes increased bacterial clearance during low-level infection, intracellular containment during high-level infection, and minimizes host cytotoxicity. In the presence of vitamin D3 host cell production of cytokines and effector molecules was infection-level dependent, most notably IL-12, which increased during high-level infection and decreased during low-level infection, and NO, which had a rate of change positively correlated to IL-12. Our study provides evidence that vitamin D3 modulation is context-dependent and time-variant, as well as highly correlated to level of infection. This study furthers our mechanistic understanding of the dual role of vitamin D3 as a regulator of bactericidal molecules and protective agent against host cell damage.

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

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.

Oxygen Modulates the Effectiveness of Granuloma Mediated Host Response to Mycobacterium tuberculosis: A Multiscale Computational Biology Approach.

Mycobacterium tuberculosis associated granuloma formation can be viewed as a structural immune response that can contain and halt the spread of the pathogen. In several mammalian hosts, including non-human primates, Mtb granulomas are often hypoxic, although this has not been observed in wild type murine infection models. While a presumed consequence, the structural contribution of the granuloma to oxygen limitation and the concomitant impact on Mtb metabolic viability and persistence remains to be fully explored. We develop a multiscale computational model to test to what extent in vivo Mtb granulomas become hypoxic, and investigate the effects of hypoxia on host immune response efficacy and mycobacterial persistence. Our study integrates a physiological model of oxygen dynamics in the extracellular space of alveolar tissue, an agent-based model of cellular immune response, and a systems biology-based model of Mtb metabolic dynamics. Our theoretical studies suggest that the dynamics of granuloma organization mediates oxygen availability and illustrates the immunological contribution of this structural host response to infection outcome. Furthermore, our integrated model demonstrates the link between structural immune response and mechanistic drivers influencing Mtbs adaptation to its changing microenvironment and the qualitative infection outcome scenarios of clearance, containment, dissemination, and a newly observed theoretical outcome of transient containment. We observed hypoxic regions in the containment granuloma similar in size to granulomas found in mammalian in vivo models of Mtb infection. In the case of the containment outcome, our model uniquely demonstrates that immune response mediated hypoxic conditions help foster the shift down of bacteria through two stages of adaptation similar to the in vitro non-replicating persistence (NRP) observed in the Wayne model of Mtb dormancy. The adaptation in part contributes to the ability of Mtb to remain dormant for years after initial infection.

Multiscale Modeling in the Clinic: Drug Design and Development.

A wide range of length and time scales are relevant to pharmacology, especially in drug development, drug design and drug delivery. Therefore, multiscale computational modeling and simulation methods and paradigms that advance the linkage of phenomena occurring at these multiple scales have become increasingly important. Multiscale approaches present in silico opportunities to advance laboratory research to bedside clinical applications in pharmaceuticals research. This is achievable through the capability of modeling to reveal phenomena occurring across multiple spatial and temporal scales, which are not otherwise readily accessible to experimentation. The resultant models, when validated, are capable of making testable predictions to guide drug design and delivery. In this review we describe the goals, methods, and opportunities of multiscale modeling in drug design and development. We demonstrate the impact of multiple scales of modeling in this field. We indicate the common mathematical and computational techniques employed for multiscale modeling approaches used in pharmacometric and systems pharmacology models in drug development and present several examples illustrating the current state-of-the-art models for (1) excitable systems and applications in cardiac disease; (2) stem cell driven complex biosystems; (3) nanoparticle delivery, with applications to angiogenesis and cancer therapy; (4) host-pathogen interactions and their use in metabolic disorders, inflammation and sepsis; and (5) computer-aided design of nanomedical systems. We conclude with a focus on barriers to successful clinical translation of drug development, drug design and drug delivery multiscale models.

A method for modeling oxygen diffusion in an agent-based model with application to host-pathogen infection.

This paper describes a method for incorporating a diffusion field modeling oxygen usage and dispersion in a multi-scale model of Mycobacterium tuberculosis (Mtb) infection mediated granuloma formation. We implemented this method over a floating-point field to model oxygen dynamics in host tissue during chronic phase response and Mtb persistence. The method avoids the requirement of satisfying the Courant-Friedrichs-Lewy (CFL) condition, which is necessary in implementing the explicit version of the finite-difference method, but imposes an impractical bound on the time step. Instead, diffusion is modeled by a matrix-based, steady state approximate solution to the diffusion equation. Presented in figure 1 is the evolution of the diffusion profiles of a containment granuloma over time.

In silico Kinetic Model of iNOS expression in macrophages.

Macrophages are a key component in the host innate response and are major contributors to the proinflammatory response against pathogens. One of the key players in the proinflammatory response is induced nitric oxide synthase (iNOS), an enzyme that provides the nitric oxide needed by phagocytic cells to create reactive nitrogen species, which are highly damaging to intracellular pathogens. To model the macrophage intracellular mechanism of iNOS gene expression, we use a systems biology approach to capture the dynamics of the iNOS gene expression system stimulated by bacterial lipopolysaccharide (LPS) and IFN-γ. Our simulation results agree with in vitro assays of iNOS gene expression and provide a platform for further investigating the potential impact of LPS and IFN-γ variations on macrophage effector function.