Digital twin mathematical models suggest individualized hemorrhagic shock resuscitation strategies.

BACKGROUND: Optimizing resuscitation to reduce inflammation and organ dysfunction following human trauma-associated hemorrhagic shock is a major clinical hurdle. This is limited by the short duration of pre-clinical studies and the sparsity of early data in the clinical setting. METHODS: We sought to bridge this gap by linking preclinical data in a porcine model with clinical data from patients from the Prospective, Observational, Multicenter, Major Trauma Transfusion (PROMMTT) study via a three-compartment ordinary differential equation model of inflammation and coagulation. RESULTS: The mathematical model accurately predicts physiologic, inflammatory, and laboratory measures in both the porcine model and patients, as well as the outcome and time of death in the PROMMTT cohort. Model simulation suggests that resuscitation with plasma and red blood cells outperformed resuscitation with crystalloid or plasma alone, and that earlier plasma resuscitation reduced injury severity and increased survival time. CONCLUSIONS: This workflow may serve as a translational bridge from pre-clinical to clinical studies in trauma-associated hemorrhagic shock and other complex disease settings.

Research to improve survival in patients with severe bleeding after major trauma presents many challenges. Here, we created a computer model to simulate the effects of severe bleeding. We refined this model using data from existing animal studies to ensure our simulations were accurate. We also used patient data to further refine the simulations to accurately predict which patients would live and which would not. We studied the effects of different treatment protocols on these simulated patients and show that treatment with plasma (the fluid portion of blood that helps form blood clots) and red blood cells jointly, gave better results than treatment with intravenous fluid or plasma alone. Early treatment with plasma reduced injury severity and increased survival time. This modelling approach may improve our ability to evaluate new treatments for trauma-associated bleeding and other acute conditions.

A common single nucleotide polymorphism is associated with inflammation and critical illness outcomes.

Acute inflammation is heterogeneous in critical illness and predictive of outcome. We hypothesized that genetic variability in novel, yet common, gene variants contributes to this heterogeneity and could stratify patient outcomes. We searched algorithmically for significant differences in systemic inflammatory mediators associated with any of 551,839 SNPs in one derivation (n = 380 patients with blunt trauma) and two validation (n = 75 trauma and n = 537 non-trauma patients) cohorts. This analysis identified rs10404939 in the LYPD4 gene. Trauma patients homozygous for the A allele (rs10404939AA; 27%) had different trajectories of systemic inflammation along with persistently elevated multiple organ dysfunction (MOD) indices vs. patients homozygous for the G allele (rs10404939GG; 26%). rs10404939AA homozygotes in the trauma validation cohort had elevated MOD indices, and non-trauma patients displayed more complex inflammatory networks and worse 90-day survival compared to rs10404939GG homozygotes. Thus, rs10404939 emerged as a common, broadly prognostic SNP in critical illness.

Peripheral nerve repair is associated with augmented cross-tissue inflammation following vascularized composite allotransplantation.

Introduction: Vascularized composite allotransplantation (VCA), with nerve repair/coaptation (NR) and tacrolimus (TAC) immunosuppressive therapy, is used to repair devastating traumatic injuries but is often complicated by inflammation spanning multiple tissues. We identified the parallel upregulation of transcriptional pathways involving chemokine signaling, T-cell receptor signaling, Th17, Th1, and Th2 pathways in skin and nerve tissue in complete VCA rejection compared to baseline in 7 human hand transplants and defined increasing complexity of protein-level dynamic networks involving chemokine, Th1, and Th17 pathways as a function of rejection severity in 5 of these patients. We next hypothesized that neural mechanisms may regulate the complex spatiotemporal evolution of rejection-associated inflammation post-VCA. Methods: For mechanistic and ethical reasons, protein-level inflammatory mediators in tissues from Lewis rats (8 per group) receiving either syngeneic (Lewis) or allogeneic (Brown-Norway) orthotopic hind limb transplants in combination with TAC, with and without sciatic NR, were compared to human hand transplant samples using computational methods. Results: In cross-correlation analyses of these mediators, VCA tissues from human hand transplants (which included NR) were most similar to those from rats undergoing VCA + NR. Based on dynamic hypergraph analyses, NR following either syngeneic or allogeneic transplantation in rats was associated with greater trans-compartmental localization of early inflammatory mediators vs. no-NR, and impaired downregulation of mediators including IL-17A at later times. Discussion: Thus, NR, while considered necessary for restoring graft function, may also result in dysregulated and mis-compartmentalized inflammation post-VCA and therefore necessitate mitigation strategies. Our novel computational pipeline may also yield translational, spatiotemporal insights in other contexts.

Inferring Tissue-Specific, TLR4-Dependent Type 17 Immune Interactions in Experimental Trauma/Hemorrhagic Shock and Resuscitation Using Computational Modeling.

Trauma/hemorrhagic shock followed by resuscitation (T/HS-R) results in multi-system inflammation and organ dysfunction, in part driven by binding of damage-associated molecular pattern molecules to Toll-like Receptor 4 (TLR4). We carried out experimental T/HS-R (pseudo-fracture plus 2 h of shock followed by 0-22 h of resuscitation) in C57BL/6 (wild type [WT]) and TLR4-null (TLR4-/-) mice, and then defined the dynamics of 20 protein-level inflammatory mediators in the heart, gut, lung, liver, spleen, kidney, and systemic circulation. Cross-correlation and Principal Component Analysis (PCA) on data from the 7 tissues sampled suggested that TLR4-/- samples express multiple inflammatory mediators in a small subset of tissue compartments as compared to the WT samples, in which many inflammatory mediators were localized non-specifically to nearly all compartments. We and others have previously defined a central role for type 17 immune cells in human trauma. Accordingly, correlations between IL-17A and GM-CSF (indicative of pathogenic Th17 cells); between IL-17A and IL-10 (indicative of non-pathogenic Th17 cells); and IL-17A and TNF (indicative of memory/effector T cells) were assessed across all tissues studied. In both WT and TLR4-/- mice, positive correlations were observed between IL-17A and GM-CSF, IL-10, and TNF in the kidney and gut. In contrast, the variable and dynamic presence of both pathogenic and non-pathogenic Th17 cells was inferred in the systemic circulation of TLR4-/- mice over time, suggesting a role for TLR4 in efflux of these cells into peripheral tissues. Hypergraph analysis - used to define dynamic, cross compartment networks - in concert with PCA-suggested that IL-17A was present persistently in all tissues at all sampled time points except for its absence in the plasma at 0.5h in the WT group, supporting the hypothesis that T/HS-R induces efflux of Th17 cells from the circulation and into specific tissues. These analyses suggest a complex, context-specific role for TLR4 and type 17 immunity following T/HS-R.

The Effects of Tacrolimus on Tissue-Specific, Protein-Level Inflammatory Networks in Vascularized Composite Allotransplantation.

Systems-level insights into inflammatory events after vascularized composite allotransplantation (VCA) are critical to the success of immunomodulatory strategies of these complex procedures. To date, the effects of tacrolimus (TAC) immunosuppression on inflammatory networks in VCA, such as in acute rejection (AR), have not been investigated. We used a systems biology approach to elucidate the effects of tacrolimus on dynamic networks and principal drivers of systemic inflammation in the context of dynamic tissue-specific immune responses following VCA. Lewis (LEW) rat recipients received orthotopic hind limb VCA from fully major histocompatibility complex-mismatched Brown Norway (BN) donors or matched LEW donors. Group 1 (syngeneic controls) received LEW limbs without TAC, and Group 2 (treatment group) received BN limbs with TAC. Time-dependent changes in 27 inflammatory mediators were analyzed in skin, muscle, and peripheral blood using Principal Component Analysis (PCA), Dynamic Bayesian Network (DyBN) inference, and Dynamic Network Analysis (DyNA) to define principal characteristics, central nodes, and putative feedback structures of systemic inflammation. Analyses were repeated on skin + muscle data to construct a "Virtual VCA", and in skin + muscle + peripheral blood data to construct a "Virtual Animal." PCA, DyBN, and DyNA results from individual tissues suggested important roles for leptin, VEGF, various chemokines, the NLRP3 inflammasome (IL-1ß, IL-18), and IL-6 after TAC treatment. The chemokines MCP-1, MIP-1α; and IP-10 were associated with AR in controls. Statistical analysis suggested that 24/27 inflammatory mediators were altered significantly between control and TAC-treated rats in peripheral blood, skin, and/or muscle over time. "Virtual VCA" and "Virtual Animal" analyses implicated the skin as a key control point of dynamic inflammatory networks, whose connectivity/complexity over time exhibited a U-shaped trajectory and was mirrored in the systemic circulation. Our study defines the effects of TAC on complex spatiotemporal evolution of dynamic inflammation networks in VCA. We also demonstrate the potential utility of computational analyses to elucidate nonlinear, cross-tissue interactions. These approaches may help define precision medicine approaches to better personalize TAC immunosuppression in VCA recipients.

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 Derivation of Core, Dynamic Human Blunt Trauma Inflammatory Endotypes.

Systemic inflammation ensues following traumatic injury, driving immune dysregulation and multiple organ dysfunction (MOD). While a balanced immune/inflammatory response is ideal for promoting tissue regeneration, most trauma patients exhibit variable and either overly exuberant or overly damped responses that likely drive adverse clinical outcomes. We hypothesized that these inflammatory phenotypes occur in the context of severe injury, and therefore sought to define clinically distinct endotypes of trauma patients based on their systemic inflammatory responses. Using Patient-Specific Principal Component Analysis followed by unsupervised hierarchical clustering of circulating inflammatory mediators obtained in the first 24 h after injury, we segregated a cohort of 227 blunt trauma survivors into three core endotypes exhibiting significant differences in requirement for mechanical ventilation, duration of ventilation, and MOD over 7 days. Nine non-survivors co-segregated with survivors. Dynamic network inference, Fisher Score analysis, and correlations of IL-17A with GM-CSF, IL-10, and IL-22 in the three survivor sub-groups suggested a role for type 3 immunity, in part regulated by Th17 and γδ 17 cells, and related tissue-protective cytokines as a key feature of systemic inflammation following injury. These endotypes may represent archetypal adaptive, over-exuberant, and overly damped inflammatory responses.

Quality Control Measures and Validation in Gene Association Studies: Lessons for Acute Illness.

Acute illness is a complex constellation of responses involving dysregulated inflammatory and immune responses, which are ultimately associated with multiple organ dysfunction. Gene association studies have associated single-nucleotide polymorphisms (SNPs) with clinical and pharmacological outcomes in a variety of disease states, including acute illness. With approximately 4 to 5 million SNPs in the human genome and recent studies suggesting that a large portion of SNP studies are not reproducible, we suggest that the ultimate clinical utility of SNPs in acute illness depends on validation and quality control measures. To investigate this issue, in December 2018 and January 2019 we searched the literature for peer-reviewed studies reporting data on associations between SNPs and clinical outcomes and between SNPs and pharmaceuticals (i.e., pharmacogenomics) published between January 2011 to February 2019. We review key methodologies and results from a variety of clinical and pharmacological gene association studies, including trauma and sepsis studies, as illustrative examples on current SNP association studies. In this review article, we have found three key points which strengthen the potential accuracy of SNP association studies in acute illness and other diseases: providing evidence of following a protocol quality control method such as the one in Nature Protocols or the OncoArray QC Guidelines; enrolling enough patients to have large cohort groups; and validating the SNPs using an independent technique such as a second study using the same SNPs with new patient cohorts. Our survey suggests the need to standardize validation methods and SNP quality control measures in medicine in general, and specifically in the context of complex disease states such as acute illness.

An Aging-Related Single-Nucleotide Polymorphism is Associated With Altered Clinical Outcomes and Distinct Inflammatory Profiles in Aged Blunt Trauma Patients.

The contribution of individual genetic determinants of aging to the adverse clinical outcomes and altered inflammation mediator networks characteristic of aged trauma patients is unknown. The AA genotype of the aging-related single-nucleotide polymorphism (SNP) rs2075650 in TOMM40 has been associated with longevity, while the AG and GG genotypes are associated with an increased risk of Alzheimer disease. Here, we studied the effect of rs2075650 on clinical outcomes and dynamic biomarker patterns after traumatic injury. Genomic DNA was obtained from blunt trauma patients admitted to the ICU and examined for 551,839 SNPs using an Illumina microarray kit. Plasma was sampled from each patient three times within the first 24 h and daily from day 1 to 7 then assayed for 31 biomarkers using Luminex. Aged patients (65-90 years) were segregated into AA (n = 77) and AG/GG (n = 17) genotypes. Additional comparisons were made with matched groups of young patients (18-30 years), controlling for injury severity score (ISS) and sex ratio, and also segregated into AA (n = 56) and AG/GG (n = 19) genotypes. Aged patients with the AA genotype had a significantly lower requirement for ventilation and fewer days on mechanical ventilation, as well as significantly higher levels of one mediator and lower levels of two mediators. Dynamic Bayesian Network inference revealed IL-23 as a central node in each network regardless of age or genotype, with MIG and IP-10 also as key mediators in the networks of the aged patients. These findings suggest that an aging-related SNP, rs2075650, may influence clinical outcomes and inflammation networks in aged patients following blunt trauma, and thus may serve as a predictive outcome biomarker in the setting of polytrauma.

MPPED2 Polymorphism Is Associated With Altered Systemic Inflammation and Adverse Trauma Outcomes.

Trauma is a leading cause of morbidity and mortality. It is unclear why some trauma victims follow a complicated clinical course and die, while others, with apparently similar injury characteristics, do not. Interpatient genomic differences, in the form of single nucleotide polymorphisms (SNPs), have been associated previously with adverse outcomes after trauma. Recently, we identified seven novel SNPs associated with mortality following trauma. The aim of the present study was to determine if one or more of these SNPs was also associated with worse clinical outcomes and altered inflammatory trajectories in trauma survivors. Accordingly, of 413 trauma survivors, DNA samples, full blood samples, and clinical data were collected at multiple time points in the first 24 h and then daily over 7 days following hospital admission. Subsequently, single-SNP groups were created and outcomes, such as hospital length of stay (LOS), ICU LOS, and requirement for mechanical ventilation, were compared. Across a broad range of Injury Severity Scores (ISS), patients carrying the rs2065418 TT SNP in the metallophosphoesterase domain-containing 2 (MPPED2) gene exhibited higher Marshall MODScores vs. the control group of rs2065418 TG/GG patients. In patients with high-severity trauma (ISS ≥ 25, n = 94), those carrying the rs2065418 TT SNP in MPPED2 exhibited higher Marshall MODScores, longer hospital LOS (21.8 ± 2 days), a greater requirement for mechanical ventilation (9.2 ± 1.4 days on ventilator, DOV), and higher creatinine plasma levels over 7 days vs. the control group of rs2065418 TG/GG high-severity trauma patients (LOS: 15.9 ± 1.2 days, p = 0.03; DOV: 5.7 ± 1 days, p = 0.04; plasma creatinine; p < 0.0001 MODScore: p = 0.0003). Furthermore, rs2065418 TT patients with ISS ≥ 25 had significantly different plasma levels of nine circulating inflammatory mediators and elevated dynamic network complexity. These studies suggest that the rs2065418 TT genotype in the MPPED2 gene is associated with altered systemic inflammation, increased organ dysfunction, and greater hospital resource utilization. A screening for this specific SNP at admission might stratify severely injured patients regarding their lung and kidney function and clinical complications.

An Enrichment Strategy Yields Seven Novel Single Nucleotide Polymorphisms Associated With Mortality and Altered Th17 Responses Following Blunt Trauma.

Trauma is the leading cause of death worldwide for individuals under the age of 55. Interpatient genomic differences, in the form of candidate single-nucleotide polymorphisms (SNPs), have been associated previously with adverse outcomes after trauma. However, the utility of these SNPs to predict outcomes based on a meaningful endpoint such as survival is as yet undefined. We hypothesized that specific SNP haplotypes could segregate trauma survivors from non-survivors. Genomic DNA samples were obtained from 453 blunt trauma patients, for whom complete daily clinical and biomarker data were available for 397. Of these, 13 patients were non-survivors and the remaining 384 were survivors. All 397 DNA samples were amplified, fragmented, and examined for 551,839 SNPs using the Illumina Infinium CoreExome-24 v1.1 BeadChip (Illumina). To enrich for likely important SNPs, we initially compared SNPs of the 13 non-survivors versus 13 matched survivors, who were matched algorithmically for injury severity score (ISS), age, and gender ratio. This initial enrichment yielded 126 SNPs; a further comparison to the haplotypes of the remaining 371 survivors yielded a final total of 7 SNPs that distinguished survivors from non-survivors. Furthermore, severely injured survivors with the same seven SNPs as non-survivor exhibited distinct inflammatory responses from similarly injured survivors without those SNPs, and specifically had evidence of altered Th17 cell phenotypes based on computational modeling. These studies suggest an interaction among genetic polymorphism, injury severity, and initial inflammatory responses in driving trauma outcomes.

"Thinking" vs. "Talking": Differential Autocrine Inflammatory Networks in Isolated Primary Hepatic Stellate Cells and Hepatocytes under Hypoxic Stress.

We hypothesized that isolated primary mouse hepatic stellate cells (HSC) and hepatocytes (HC) would elaborate different inflammatory responses to hypoxia with or without reoxygenation. We further hypothesized that intracellular information processing ("thinking") differs from extracellular information transfer ("talking") in each of these two liver cell types. Finally, we hypothesized that the complexity of these autocrine responses might only be defined in the absence of other non-parenchymal cells or trafficking leukocytes. Accordingly, we assayed 19 inflammatory mediators in the cell culture media (CCM) and whole cell lysates (WCLs) of HSC and HC during hypoxia with and without reoxygenation. We applied a unique set of statistical and data-driven modeling techniques including Two-Way ANOVA, hierarchical clustering, Principal Component Analysis (PCA) and Network Analysis to define the inflammatory responses of these isolated cells to stress. HSC, under hypoxic and reoxygenation stresses, both expressed and secreted larger quantities of nearly all inflammatory mediators as compared to HC. These differential responses allowed for segregation of HSC from HC by hierarchical clustering. PCA suggested, and network analysis supported, the hypothesis that above a certain threshold of cellular stress, the inflammatory response becomes focused on a limited number of functions in both HSC and HC, but with distinct characteristics in each cell type. Network analysis of separate extracellular and intracellular inflammatory responses, as well as analysis of the combined data, also suggested the presence of more complex inflammatory "talking" (but not "thinking") networks in HSC than in HC. This combined network analysis also suggested an interplay between intracellular and extracellular mediators in HSC under more conditions than that observed in HC, though both cell types exhibited a qualitatively similar phenotype under hypoxia/reoxygenation. Our results thus suggest that a stepwise series of computational and statistical analyses may help decipher how cells respond to environmental stresses, both within the cell and in its secretory products, even in the absence of cooperation from other cells in the liver.