The genetic background shapes the susceptibility to mitochondrial dysfunction and NASH progression.

Non-alcoholic steatohepatitis (NASH) is a global health concern without treatment. The challenge in finding effective therapies is due to the lack of good mouse models and the complexity of the disease, characterized by gene-environment interactions. We tested the susceptibility of seven mouse strains to develop NASH. The severity of the clinical phenotypes observed varied widely across strains. PWK/PhJ mice were the most prone to develop hepatic inflammation and the only strain to progress to NASH with extensive fibrosis, while CAST/EiJ mice were completely resistant. Levels of mitochondrial transcripts and proteins as well as mitochondrial function were robustly reduced specifically in the liver of PWK/PhJ mice, suggesting a central role of mitochondrial dysfunction in NASH progression. Importantly, the NASH gene expression profile of PWK/PhJ mice had the highest overlap with the human NASH signature. Our study exposes the limitations of using a single mouse genetic background in metabolic studies and describes a novel NASH mouse model with features of the human NASH.

Mitochondrial and NAD+ metabolism predict recovery from acute kidney injury in a diverse mouse population.

Acute kidney failure and chronic kidney disease are global health issues steadily rising in incidence and prevalence. Animal models on a single genetic background have so far failed to recapitulate the clinical presentation of human nephropathies. Here, we used a simple model of folic acid-induced kidney injury in 7 highly diverse mouse strains. We measured plasma and urine parameters, as well as renal histopathology and mRNA expression data, at 1, 2, and 6 weeks after injury, covering the early recovery and long-term remission. We observed an extensive strain-specific response ranging from complete resistance of the CAST/EiJ to high sensitivity of the C57BL/6J, DBA/2J, and PWK/PhJ strains. In susceptible strains, the severe early kidney injury was accompanied by the induction of mitochondrial stress response (MSR) genes and the attenuation of NAD+ synthesis pathways. This is associated with delayed healing and a prolonged inflammatory and adaptive immune response 6 weeks after insult, heralding a transition to chronic kidney disease. Through a thorough comparison of the transcriptomic response in mouse and human disease, we show that critical metabolic gene alterations were shared across species, and we highlight the PWK/PhJ strain as an emergent model of transition from acute kidney injury to chronic disease.

A putative "chemokine switch" that regulates systemic acute inflammation in humans.

Systemic inflammation is complex and likely drives clinical outcomes in critical illness such as that which ensues following severe injury. We obtained time course data on multiple inflammatory mediators in the blood of blunt trauma patients. Using dynamic network analyses, we inferred a novel control architecture for systemic inflammation: a three-way switch comprising the chemokines MCP-1/CCL2, MIG/CXCL9, and IP-10/CXCL10. To test this hypothesis, we created a logical model comprising this putative architecture. This model predicted key qualitative features of systemic inflammation in patient sub-groups, as well as the different patterns of hospital discharge of moderately vs. severely injured patients. Thus, a rational transition from data to data-driven models to mechanistic models suggests a novel, chemokine-based mechanism for control of acute inflammation in humans and points to the potential utility of this workflow in defining novel features in other complex diseases.

Computational Analysis Supports an Early, Type 17 Cell-Associated Divergence of Blunt Trauma Survival and Mortality.

OBJECTIVE: Blunt trauma patients may present with similar demographics and injury severity yet differ with regard to survival. We hypothesized that this divergence was due to different trajectories of systemic inflammation and utilized computational analyses to define these differences. DESIGN: Retrospective clinical study and experimental study in mice. SETTING: Level 1 trauma center and experimental laboratory. PATIENTS: From a cohort of 493 victims of blunt trauma, we conducted a pairwise, retrospective, case-control study of patients who survived over 24 hours but ultimately died (nonsurvivors; n = 19) and patients who, after ICU admission, went on to be discharged(survivors; n = 19). INTERVENTIONS: None in patients. Neutralizing anti-interleukin-17A antibody in mice. MEASUREMENTS AND MAIN RESULTS: Data on systemic inflammatory mediators assessed within the first 24 hours and over 7 days were analyzed with computational modeling to infer dynamic networks of inflammation. Network density among inflammatory mediators in nonsurvivors increased in parallel with organ dysfunction scores over 7 days, suggesting the presence of early, self-sustaining, pathologic inflammation involving high-mobility group protein B1, interleukin-23, and the Th17 pathway. Survivors demonstrated a pattern commensurate with a self-resolving, predominantly lymphoid response, including higher levels of the reparative cytokine interleukin-22. Mice subjected to trauma/hemorrhage exhibited reduced organ damage when treated with anti-interleukin-17A. CONCLUSIONS: Variable type 17 immune responses are hallmarks of organ damage, survival, and mortality after blunt trauma and suggest a lymphoid cell-based switch from self-resolving to self-sustaining inflammation.

Insights into the Role of Chemokines, Damage-Associated Molecular Patterns, and Lymphocyte-Derived Mediators from Computational Models of Trauma-Induced Inflammation.

SIGNIFICANCE: Traumatic injury elicits a complex, dynamic, multidimensional inflammatory response that is intertwined with complications such as multiple organ dysfunction and nosocomial infection. The complex interplay between inflammation and physiology in critical illness remains a challenge for translational research, including the extrapolation to human disease from animal models. RECENT ADVANCES: Over the past decade, we and others have attempted to decipher the biocomplexity of inflammation in these settings of acute illness, using computational models to improve clinical translation. In silico modeling has been suggested as a computationally based framework for integrating data derived from basic biology experiments as well as preclinical and clinical studies. CRITICAL ISSUES: Extensive studies in cells, mice, and human blunt trauma patients have led us to suggest (i) that while an adequate level of inflammation is required for healing post-trauma, inflammation can be harmful when it becomes self-sustaining via a damage-associated molecular pattern/Toll-like receptor-driven feed-forward circuit; (ii) that chemokines play a central regulatory role in driving either self-resolving or self-maintaining inflammation that drives the early activation of both classical innate and more recently recognized lymphoid pathways; and (iii) the presence of multiple thresholds and feedback loops, which could significantly affect the propagation of inflammation across multiple body compartments. FUTURE DIRECTIONS: These insights from data-driven models into the primary drivers and interconnected networks of inflammation have been used to generate mechanistic computational models. Together, these models may be used to gain basic insights as well as serving to help define novel biomarkers and therapeutic targets.

In vivo and systems biology studies implicate IL-18 as a central mediator in chronic pain.

Inflammation is associated with peripheral neuropathy, however the interplay among cytokines, chemokines, and neurons is still unclear. We hypothesized that this neuroinflammatory interaction can be defined by computational modeling based on the dynamics of protein expression in the sciatic nerve of rats subjected to chronic constriction injury. Using Dynamic Bayesian Network inference, we identified interleukin (IL)-18 as a central node associated with neuropathic pain in this animal model. Immunofluorescence supported a role for inflammasome activation and induction of IL-18 at the site of injury. Combined in vivo and in silico approaches may thus highlight novel targets in peripheral neuropathy.

Innate immunity in disease: insights from mathematical modeling and analysis.

The acute inflammatory response is a complex defense mechanism that has evolved to respond rapidly to injury, infection, and other disruptions in homeostasis. This robust responsiveness to biological stress likely endows the host with increased fitness, but over-robust or inadequate inflammation predisposes the host to various diseases. Importantly, well-compartmentalized inflammation is generally beneficial, but spillover of inflammation into the blood is a hallmark-and likely also a driver-of self-maintaining inflammation. The blood is also a key entry point and immunological interface for vectors of parasitic diseases, diseases that themselves incite systemic inflammation. The complex role of inflammation in health and disease has made this biological system difficult to understand comprehensively and modulate rationally for therapeutic purposes. Consequently, systems approaches have been applied in order to characterize dynamical properties and identify key control points in inflammation. This process begins with the collection of high-dimensional, experimental, and clinical data, followed by data reduction and data-driven modeling that finally informs mechanistic computational models for analysis, prediction, and rational modulation. These studies have suggested that the overall architecture of the inflammatory response includes a multiscale positive feedback from inflammation → tissue damage → inflammation, which is often inadequately controlled by negative feedback from anti-inflammatory mediators. Given the importance of the blood interface for the inflammatory response, and the accessibility of this compartment both as an immunological sampling reservoir for vectors as well as for diagnosis and therapy, we suggest that any rational efforts at modulating inflammation via the blood compartment must involve computational modeling.

Inducible protein-10, a potential driver of neurally controlled interleukin-10 and morbidity in human blunt trauma.

OBJECTIVE: Blunt trauma and traumatic spinal cord injury induce systemic inflammation that contributes to morbidity. Dysregulated neural control of systemic inflammation postinjury is likely exaggerated in patients with traumatic spinal cord injury. We used in silico methods to discern dynamic inflammatory networks that could distinguish systemic inflammation in traumatic spinal cord injury from blunt trauma. DESIGN: Retrospective study. SETTINGS: Tertiary care institution. PATIENTS: Twenty-one severely injured thoracocervical traumatic spinal cord injury patients and matched 21 severely injured blunt trauma patients without spinal cord injury. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Serial blood samples were obtained from days 1 to 14 postinjury. Twenty-four plasma inflammatory mediators were quantified. Statistical significance between the two groups was determined by two-way analysis of variance. Dynamic Bayesian network inference was used to suggest dynamic connectivity and central inflammatory mediators. Circulating interleukin-10 was significantly elevated in thoracocervical traumatic spinal cord injury group versus non-spinal cord injury group, whereas interleukin-1ß, soluble interleukin-2 receptor-α, interleukin-4, interleukin-5, interleukin-7, interleukin-13, interleukin-17, macrophage inflammatory protein 1α and 1ß, granulocyte-macrophage colony-stimulating factor, and interferon-γ were significantly reduced in traumatic spinal cord injury group versus non-spinal cord injury group. Dynamic Bayesian network suggested that post-spinal cord injury interleukin-10 is driven by inducible protein-10, whereas monocyte chemotactic protein-1 was central in non-spinal cord injury dynamic networks. In a separate validation cohorts of 356 patients without spinal cord injury and 85 traumatic spinal cord injury patients, individuals with plasma inducible protein-10 levels more than or equal to 730 pg/mL had significantly prolonged hospital and ICU stay and days on mechanical ventilator versus patients with plasma inducible protein-10 level less than 730 pg/mL. CONCLUSION: This is the first study to compare the dynamic systemic inflammatory responses of traumatic spinal cord injury patients versus patients without spinal cord injury, suggesting a key role for inducible protein-10 in driving systemic interleukin-10 and morbidity and highlighting the potential utility of in silico tools to identify key inflammatory drivers.

Removal of inflammatory ascites is associated with dynamic modification of local and systemic inflammation along with prevention of acute lung injury: in vivo and in silico studies.

BACKGROUND: Sepsis-induced inflammation in the gut/peritoneal compartment occurs early in sepsis and can lead to acute lung injury (ALI). We have suggested that inflammatory ascites drives the pathogenesis of ALI and that removal of ascites with an abdominal wound vacuum prevents ALI. We hypothesized that the time- and compartment-dependent changes in inflammation that determine this process can be discerned using principal component analysis (PCA) and Dynamic Bayesian Network (DBN) inference. METHODS: To test this hypothesis, data from a previous study were analyzed using PCA and DBN. In that study, two groups of anesthetized, ventilated pigs were subjected to experimental sepsis via intestinal ischemia/reperfusion and placement of a peritoneal fecal clot. The control group (n = 6) had the abdomen opened at 12 h after injury (T12) with attachment of a passive drain. The peritoneal suction treatment (PST) group (n = 6) was treated in an identical fashion except that a vacuum was applied to the peritoneal cavity at T12 to remove ascites and maintained until T48. Multiple inflammatory mediators were measured in ascites and plasma and related to lung function (PaO2/FIO2 ratio and oxygen index) using PCA and DBN. RESULTS: Peritoneal suction treatment prevented ALI based on lung histopathology, whereas control animals developed ALI. Principal component analysis revealed that local to the insult (i.e., ascites), primary proinflammatory cytokines play a decreased role in the overall response in the treatment group as compared with control. In both groups, multiple, nested positive feedback loops were inferred from DBN, which included interrelated roles for bacterial endotoxin, interleukin 6, transforming growth factor ß1, C-reactive protein, PaO2/FIO2 ratio, and oxygen index. von Willebrand factor was an output in control, but not PST, ascites. CONCLUSIONS: These combined in vivo and in silico studies suggest that in this clinically realistic paradigm of sepsis, endotoxin drives the inflammatory response in the ascites, interplaying with lung dysfunction in a feed-forward loop that exacerbates inflammation and leads to endothelial dysfunction, systemic spillover, and ALI; PST partially modifies this process.

Analysis of serum inflammatory mediators identifies unique dynamic networks associated with death and spontaneous survival in pediatric acute liver failure.

BACKGROUND: Tools to predict death or spontaneous survival are necessary to inform liver transplantation (LTx) decisions in pediatric acute liver failure (PALF), but such tools are not available. Recent data suggest that immune/inflammatory dysregulation occurs in the setting of acute liver failure. We hypothesized that specific, dynamic, and measurable patterns of immune/inflammatory dysregulation will correlate with outcomes in PALF. METHODS: We assayed 26 inflammatory mediators on stored serum samples obtained from a convenience sample of 49 children in the PALF study group (PALFSG) collected within 7 days after enrollment. Outcomes were assessed within 21 days of enrollment consisting of spontaneous survivors, non-survivors, and LTx recipients. Data were subjected to statistical analysis, patient-specific Principal Component Analysis (PCA), and Dynamic Bayesian Network (DBN) inference. FINDINGS: Raw inflammatory mediator levels assessed over time did not distinguish among PALF outcomes. However, DBN analysis did reveal distinct interferon-gamma-related networks that distinguished spontaneous survivors from those who died. The network identified in LTx patients pre-transplant was more like that seen in spontaneous survivors than in those who died, a finding supported by PCA. INTERPRETATION: The application of DBN analysis of inflammatory mediators in this small patient sample appears to differentiate survivors from non-survivors in PALF. Patterns associated with LTx pre-transplant were more like those seen in spontaneous survivors than in those who died. DBN-based analyses might lead to a better prediction of outcome in PALF, and could also have more general utility in other complex diseases with an inflammatory etiology.

In vivo, in vitro, and in silico studies suggest a conserved immune module that regulates malaria parasite transmission from mammals to mosquitoes.

Human malaria can be caused by the parasite Plasmodium falciparum that is transmitted by female Anopheles mosquitoes. "Immunological crosstalk" between the mammalian and anopheline hosts for Plasmodium functions to control parasite numbers. Key to this process is the mammalian cytokine transforming growth factor-ß1 (TGF-ß1). In mammals, TGF-ß1 regulates inducible nitric oxide (NO) synthase (iNOS) both positively and negatively. In some settings, high levels of NO activate latent TGF-ß1, which in turn suppresses iNOS expression. In the mosquito, ingested TGF-ß1 induces A. stephensi NOS (AsNOS), which limits parasite development and which in turn is suppressed by activation of the mosquito homolog of the mitogen-activated protein kinases MEK and ERK. Computational models linking TGF-ß1, AsNOS, and MEK/ERK were developed to provide insights into this complex biology. An initial Boolean model suggested that, as occurs in mammalian cells, MEK/ERK and AsNOS would oscillate upon ingestion of TGF-ß1. An ordinary differential equation (ODE) model further supported the hypothesis of TGF-ß1-induced multiphasic behavior of MEK/ERK and AsNOS. To achieve this multiphasic behavior, the ODE model was predicated on the presence of constant levels of TGF-ß1 in the mosquito midgut. Ingested TGF-ß1, however, did not exhibit this behavior. Accordingly, we hypothesized and experimentally verified that ingested TGF-ß1 induces the expression of the endogenous mosquito TGF-ß superfamily ligand As60A. Computational simulation of these complex, cross-species interactions suggested that TGF-ß1 and NO-mediated induction of As60A expression together may act to maintain multiphasic AsNOS expression via MEK/ERK-dependent signaling. We hypothesize that multiphasic behavior as represented in this model allows the mosquito to balance the conflicting demands of parasite killing and metabolic homeostasis in the face of damaging inflammation.

Identification of a novel pathway of transforming growth factor-ß1 regulation by extracellular NAD+ in mouse macrophages: in vitro and in silico studies.

Extracellular ß-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-ß1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-ß1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-ß1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-ß1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-ß1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-ß1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-ß1.

Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages.

Milk fat globule epidermal growth factor 8 (Mfge8) is a soluble glycoprotein known to regulate inflammation and immunity by mediating apoptotic cell clearance. Since fibrosis can occur as a result of exaggerated apoptosis and inflammation, we set out to investigate the hypothesis that Mfge8 might negatively regulate tissue fibrosis. We report here that Mfge8 does decrease the severity of tissue fibrosis in a mouse model of pulmonary fibrosis; however, it does so not through effects on inflammation and apoptotic cell clearance, but by binding and targeting collagen for cellular uptake through its discoidin domains. Initial analysis revealed that Mfge8-/- mice exhibited enhanced pulmonary fibrosis after bleomycin-induced lung injury. However, they did not have increased inflammation or impaired apoptotic cell clearance after lung injury compared with Mfge8+/+ mice; rather, they had a defect in collagen turnover. Further experiments indicated that Mfge8 directly bound collagen and that Mfge8-/- macrophages exhibited defective collagen uptake that could be rescued by recombinant Mfge8 containing at least one discoidin domain. These data demonstrate a critical role for Mfge8 in decreasing the severity of murine tissue fibrosis by facilitating the removal of accumulated collagen.