Implications of an Altered Gut Microbiome in Rat Experimental Vascularized Composite Transplantation.

OBJECTIVES: Vascularized composite allotransplantation is a reconstructive option after severe injury but is fraught with complications, including transplant rejection due to major histocompatibility complex mismatch in the context of allogeneic transplant, which in turn is due to altered immuno-inflammation secondary to transplant. The immunosuppressant tacrolimus can prevent rejection. Because tacrolimus is metabolized predominantly by the gut, this immunosuppressant alters the gut microbiome in multiple ways, thereby possibly affecting immunoinflammation. MATERIALS AND METHODS: We performed either allogeneic or syngeneic transplant with or without tacrolimus in rats. We quantified protein-level inflammatory mediators in the skin, muscle, and plasma and assessed the diversity of the gut microbiome through 16S RNA analysis at several timepoints over 31 days posttransplant. RESULTS: Statistical analysis highlighted a complex interaction between major histocompatibility complex and tacrolimus therapy on the relative diversity of the microbiome. Time-interval principal component analysis indicated numerous significant differences in the tissue characteristics of inflammation and gut microbiome that varied over time and across experimental conditions. Classification and regression tree analysis suggested that both inflammatory mediators in specific tissues and changes in the gut microbiome are useful in characterizing the temporal dynamics of posttransplant inflammation. Dynamic network analysis highlighted unique changes in Methanosphaera that were correlated with Peptococcusin allogeneic transplants with and without tacrolimus versus Prevotella in syngeneic transplant with tacrolimus, suggesting that alterations in Methanosphaera might be a biomarker of vascularized composite allotransplant rejection. CONCLUSIONS: Our results suggest a complex interaction among major histocompatibility complex, local and systemic immuno-inflammation, and tacrolimus therapy and highlight the potential for novel insights into vascularized composite allotransplant from computational approaches.

Unraveling the complex relationship between mRNA and protein abundances: a machine learning-based approach for imputing protein levels from RNA-seq data.

The correlation between messenger RNA (mRNA) and protein abundances has long been debated. RNA sequencing (RNA-seq), a high-throughput, commonly used method for analyzing transcriptional dynamics, leaves questions about whether we can translate RNA-seq-identified gene signatures directly to protein changes. In this study, we utilized a set of 17 widely assessed immune and wound healing mediators in the context of canine volumetric muscle loss to investigate the correlation of mRNA and protein abundances. Our data reveal an overall agreement between mRNA and protein levels on these 17 mediators when examining samples from the same experimental condition (e.g. the same biopsy). However, we observed a lack of correlation between mRNA and protein levels for individual genes under different conditions, underscoring the challenges in converting transcriptional changes into protein changes. To address this discrepancy, we developed a machine learning model to predict protein abundances from RNA-seq data, achieving high accuracy. Our approach also effectively corrected multiple extreme outliers measured by antibody-based protein assays. Additionally, this model has the potential to detect post-translational modification events, as shown by accurately estimating activated transforming growth factor ß1 levels. This study presents a promising approach for converting RNA-seq data into protein abundance and its biological significance.

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.

Baseline Plasma Inflammatory Profile Is Associated With Response to Neoadjuvant Chemotherapy in Patients With Pancreatic Adenocarcinoma.

Despite its increased application in pancreatic ductal adenocarcinoma (PDAC), complete response to neoadjuvant therapy (NAT) is rare. Given the critical role of host immunity in regulating cancer, we sought to correlate baseline inflammatory profiles to significant response to NAT. PDAC patients receiving NAT were classified as responders (R) or nonresponders (NR) by carbohydrate antigen 19-9 response, pathologic tumor size, and lymph node status in the resected specimen. Baseline (treatment-naive) plasma was analyzed to determine levels of 27 inflammatory mediators. Logistic regression was used to correlate individual mediators with response. Network analysis and Pearson correlation maps were derived to determine baseline inflammatory mediator profiles. Forty patients (20R and 20NR) met study criteria. The R showed significantly higher overall survival (59.4 vs. 21.25 mo, P=0.002) and disease-free survival (50.97 vs. 10.60 mo, P=0.005), compared with NR. soluble interleukin-2 receptor alpha was a significant predictor of no response to NAT (P=0.045). Analysis of inflammatory profiles using the Pearson heat map analysis followed by network analysis depicted increased inflammatory network complexity in NR compared with R (1.69 vs. 1), signifying a more robust baseline inflammatory status of NR. A panel of inflammatory mediators identified by logistic regression and Fischer score analysis was used to create a potential decision tree to predict NAT response. We demonstrate that baseline inflammatory profiles are associated with response to NAT in PDAC, and that an upregulated inflammatory status is associated with a poor response to NAT. Further analysis into the role of inflammatory mediators as predictors of chemotherapy response is warranted.

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.

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.

Inflammatory signals and network connections implicate cell-mediated immunity in chronic venous insufficiency.

BACKGROUND: The role of inflammation in superficial venous reflux in varicose veins (VVs) is unknown. Computational network modeling has deduced inflammation in experimental and clinical settings. We measured immune mediators in plasma from competent and incompetent leg veins inferring the role of cellular immunity based on cytokine networks. METHODS: Temperature was assessed using infrared thermography (IRT) to measure inflammation. Blood was obtained during sclerotherapy or endovenous thermal ablation for VVs. Control subjects underwent phlebotomy from saphenous and forearm veins. Vein segments were harvested during surgery. Demographics, clinical, etiology, anatomy and pathophysiology classification, venous clinical severity scores (VCSSs), and body mass index (BMI) were collected. Twenty-five mediators were measured in serum and vein segments. Means were compared using Mann-Whitney U test. Pearson correlations equaling or exceeding a threshold prompted connections among nodes, and mapped as networks. Spearman correlations were performed between interleukin (IL)-17A and both granulocyte macrophage colony stimulation factor, and IL-10 as indicators of pathogenic and nonpathogenic Th17 cell involvement. RESULTS: Age, BMI, and VCSSs differed significantly between groups. Temperatures were higher over diseased veins. Plasma concentrations of 20 cytokines differed between control and patient subjects (P<0.05), and most were lower in patients. C-X-C motif chemokine ligand-9 (aka monokine-induced by gamma interferon), C-X-C motif chemokine ligand 10 (aka IFNγ induced protein 10), and soluble IL-2 receptor-alpha were higher in patients, but not connected to other mediators in networks. In contrast, IL-17A, IL-12p70, and interferon gamma were the only mediators that were more highly interconnected in venous insufficiency. IL-17A and granulocyte macrophage colony stimulating factor (GM-CSF) were highly correlated in chronic venous insufficiency (CVI) but not in controls. In tissue, refluxing VVs significantly higher IL-15 expression than competent saphenous veins. CONCLUSIONS: Venous insufficiency associates with age, BMI, skin temperature, and plasma cytokines associated with interferon gamma and possibly IL-17A signaling. The vein wall may be a source of activation of cellular activation, given elevated IL-15 expression. Correlations between IL-17A and GM-CSF suggested a potential role for pathogenic Th17 cells in VVs. Differentially expressed inflammatory networks induced by venous hypertension may reflect or drive venous damage and ulceration.

Prehospital plasma is associated with distinct biomarker expression following injury.

BACKGROUNDPrehospital plasma improves survival in severely injured patients transported by air ambulance. We hypothesized that prehospital plasma would be associated with a reduction in immune imbalance and endothelial damage.METHODSWe sampled blood from 405 trauma patients enrolled in the Prehospital Air Medical Plasma (PAMPer) trial upon hospital admission (0 hours) and 24 hours post admission across 6 U.S. sites. We assayed samples for 21 inflammatory mediators and 7 markers associated with endothelial function and damage. We performed hierarchical clustering analysis (HCA) of these biomarkers of the immune response and endothelial injury. Regression analysis was used to control for differences across study and to assess any association with prehospital plasma resuscitation.RESULTSHCA distinguished two patient clusters with different injury patterns and outcomes. Patients in cluster A had greater injury severity and incidence of blunt trauma, traumatic brain injury, and mortality. Cluster A patients that received prehospital plasma showed improved 30-day survival. Prehospital plasma did not improve survival in cluster B patients. In an adjusted analysis of the most seriously injured patients, prehospital plasma was associated with an increase in adiponectin, IL-1ß, IL-17A, IL-23, and IL-17E upon admission, and a reduction in syndecan-1, TM, VEGF, IL-6, IP-10, MCP-1, and TNF-α, and an increase in IL-33, IL-21, IL-23, and IL-17E 24 hours later.CONCLUSIONPrehospital plasma may ameliorate immune dysfunction and the endotheliopathy of trauma. These effects of plasma may contribute to improved survival in injured patients.TRIAL REGISTRATIONNCT01818427.FUNDINGDepartment of Defense; National Institutes of Health, U.S. Army.

Dynamics of Systemic Inflammation as a Function of Developmental Stage in Pediatric Acute Liver Failure.

The Pediatric Acute Liver Failure (PALF) study is a multicenter, observational cohort study of infants and children diagnosed with this complex clinical syndrome. Outcomes in PALF reflect interactions among the child's clinical condition, response to supportive care, disease severity, potential for recovery, and, if needed, availability of a suitable organ for liver transplantation (LTx). Previously, we used computational analyses of immune/inflammatory mediators that identified three distinct dynamic network patterns of systemic inflammation in PALF associated with spontaneous survivors, non-survivors (NS), and LTx recipients. To date, there are no data exploring age-specific immune/inflammatory responses in PALF. Accordingly, we measured a number of clinical characteristics and PALF-associated systemic inflammatory mediators in daily serum samples collected over the first 7 days following enrollment from five distinct PALF cohorts (all spontaneous survivors without LTx): infants (INF, <1 year), toddlers (TOD, 1-2 years.), young children (YCH, 2-4 years), older children (OCH, 4-13 years) and adolescents (ADO, 13-18 years). Among those groups, we observed significant (P<0.05) differences in ALT, creatinine, Eotaxin, IFN-γ, IL-1RA, IL-1ß, IL-2, sIL-2Rα, IL-4, IL-6, IL-12p40, IL-12p70, IL-13, IL-15, MCP-1, MIP-1α, MIP-1ß, TNF-α, and NO2-/NO3- . Dynamic Bayesian Network inference identified a common network motif with HMGB1 as a central node in all sub-groups, with MIG/CXCL9 being a central node in all groups except INF. Dynamic Network Analysis (DyNA) inferred different dynamic patterns and overall dynamic inflammatory network complexity as follows: OCH>INF>TOD>ADO>YCH. Hypothesizing that systemically elevated but sparsely connected inflammatory mediators represent pathological inflammation, we calculated the AuCon score (area under the curve derived from multiple measures over time divided by DyNA connectivity) for each mediator, and identified HMGB1, MIG, IP-10/CXCl10, sIL-2Rα, and MCP-1/CCL2 as potential correlates of PALF pathophysiology, largely in agreement with the results of Partial Least Squares Discriminant Analysis. Since NS were in the INF age group, we compared NS to INF and found greater inflammatory coordination and dynamic network connectivity in NS vs. INF. HMGB1 was the sole central node in both INF and NS, though NS had more downstream nodes. Thus, multiple machine learning approaches were used to gain both basic and potentially translational insights into a complex inflammatory disease.

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.

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.

Diurnal Variation in Systemic Acute Inflammation and Clinical Outcomes Following Severe Blunt Trauma.

Animal studies suggest that the time of day is a determinant of the immunological response to both injury and infection. We hypothesized that due to this diurnal variation, time of injury could affect the systemic inflammatory response and outcomes post-trauma and tested this hypothesis by examining the dynamics of circulating inflammatory mediators in blunt trauma patients injured during daytime vs. nighttime. From a cohort of 472 blunt trauma survivors, two stringently matched sub-cohorts of moderately/severely injured patients [injury severity score (ISS) >20] were identified. Fifteen propensity-matched, daytime-inured ("mDay") patients (age 43.6 ± 5.2, M/F 11/4, ISS 22.9 ± 0.7) presented during the shortest local annual period (8:00 am-5:00 pm), and 15 propensity-matched "mNight" patients (age 43 ± 4.3, M/F 11/4, ISS 24.5 ± 2.5) presented during the shortest night period (10:00 pm-5:00 am). Serial blood samples were obtained (3 samples within the first 24 h and daily from days 1-7) from all patients. Thirty-two plasma inflammatory mediators were assayed. Two-way Analysis of Variance (ANOVA) was used to compare groups. Dynamic Network Analysis (DyNA) and Dynamic Bayesian Network (DyBN) inference were utilized to infer dynamic interrelationships among inflammatory mediators. Both total hospital and intensive care unit length of stay were significantly prolonged in the mNight group. Circulating IL-17A was elevated significantly in the mNight group from 24 h to 7 days post-injury. Circulating MIP-1α, IL-7, IL-15, GM-CSF, and sST2 were elevated in the mDay group. DyNA demonstrated elevated network complexity in the mNight vs. the mDay group. DyBN suggested that cortisol and sST2 were central nodes upstream of TGF-ß1, chemokines, and Th17/protective mediators in both groups, with IL-6 being an additional downstream node in the mNight group only. Our results suggest that time of injury affects clinical outcomes in severely injured patients in a manner associated with an altered systemic inflammation program, possibly implying a role for diurnal or circadian variation in the response to traumatic injury.

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.

Decoding the secreted inflammatory response of primary human hepatocytes to hypoxic stress in vitro.

BACKGROUND: The cellular and molecular response of liver cells to hypoxic stress is not fully understood. We used computational modeling to gain insights into the inflammatory response of primary human hepatocytes (HC) to hypoxic stress in vitro. METHODS: Primary HC from cancer patients were exposed to hypoxia (1% O2) or normoxia (21% O2) for 1-48 h, and the cell supernatants were assayed for 21 inflammatory mediators. Data were analyzed by Two-Way ANOVA, Dynamic Bayesian Network (DBN) inference, Dynamic Network Analysis (DyNA), and Time-interval Principal Component Analysis (TI-PCA). RESULTS: The chemokines MCP-1/CCL2 and IP-10/CXCL10, along with the cytokines interleukin (IL)-2 and IL-15 were altered significantly over time in hypoxic vs. normoxic HC. DBN inference suggested central, coordinating roles for MCP-1 and IL-8 in regulating a largely conserved inflammatory program in both hypoxic and normoxic HC. DyNA likewise suggested similar network trajectories of decreasing complexity over time in both hypoxic and normoxic HC, though with differential connectivity of MCP-1, IP-10, IL-8, and Eotaxin. TI-PCA pointed to IL-1ß as a central characteristic of inflammation in hypoxic HC across all time intervals, along with IL-15 and IL-10, vs. Eotaxin, IL-7, IL-10, IL-15, and IL-17A in normoxic HC. CONCLUSIONS: Thus, diverse human HC appear to respond in a largely conserved fashion to cell culture stress, with distinct characteristics based on the presence or absence of hypoxia.

HMGB1 is a Central Driver of Dynamic Pro-inflammatory Networks in Pediatric Acute Liver Failure induced by Acetaminophen.

Acetaminophen (APAP) overdose (APAPo) is predominant in the NIH Pediatric Acute Liver Failure (PALF) Study. We assayed multiple inflammatory mediators in serial serum samples from 13 PALF survivors with APAPo + N-acetylcysteine (NAC, the frontline therapy for APAPo), 8 non-APAPo + NAC, 40 non-APAPo non-NAC, and 12 non-survivors. High Mobility Group Box 1 (HMGB1) was a dominant mediator in dynamic inflammation networks in all sub-groups, associated with a threshold network complexity event at d1-2 following enrollment that was exceeded in non-survivors vs. survivors. We thus hypothesized that differential HMGB1 network connectivity after day 2 is related to the putative threshold event in non-survivors. DyNA showed that HMGB1 is most connected in non-survivors on day 2-3, while no connections were observed in APAPo + NAC and non-APAPo + NAC survivors. Inflammatory dynamic networks, and in particular HMGB1 connectivity, were associated with the use of NAC in the context of APAPo. To recapitulate hepatocyte (HC) damage in vitro, primary C57BL/6 HC and HC-specific HMGB1-null HC were treated with APAP + NAC. Network phenotypes of survivors were recapitulated in C57BL/6 mouse HC and were greatly altered in HMGB1-null HC. HC HMGB1 may thus coordinate a pro-inflammatory program in PALF non-survivors (which is antagonized by NAC), while driving an anti-inflammatory/repair program in survivors.

A computational analysis of dynamic, multi-organ inflammatory crosstalk induced by endotoxin in mice.

Bacterial lipopolysaccharide (LPS) induces an acute inflammatory response across multiple organs, primarily via Toll-like receptor 4 (TLR4). We sought to define novel aspects of the complex spatiotemporal dynamics of LPS-induced inflammation using computational modeling, with a special focus on the timing of pathological systemic spillover. An analysis of principal drivers of LPS-induced inflammation in the heart, gut, lung, liver, spleen, and kidney to assess organ-specific dynamics, as well as in the plasma (as an assessment of systemic spillover), was carried out using data on 20 protein-level inflammatory mediators measured over 0-48h in both C57BL/6 and TLR4-null mice. Using a suite of computational techniques, including a time-interval variant of Principal Component Analysis, we confirm key roles for cytokines such as tumor necrosis factor-α and interleukin-17A, define a temporal hierarchy of organ-localized inflammation, and infer the point at which organ-localized inflammation spills over systemically. Thus, by employing a systems biology approach, we obtain a novel perspective on the time- and organ-specific components in the propagation of acute systemic inflammation.

Suppressed networks of inflammatory mediators characterize chronic venous insufficiency.

OBJECTIVE: Chronic venous insufficiency (CVI) affects 25 million adults in the United States. Little emphasis has been placed on inflammatory changes associated with CVI. We hypothesize that in patients with early to mid-stage benign varicose vein disease, differences in circulating inflammatory mediators will be manifested in blood draining the involved area vs circulating blood in control subjects. METHODS: Patients undergoing either endovenous ablation or sclerotherapy for Clinical, Etiology, Anatomy, and Pathophysiology clinical class 3 to 5 disease underwent phlebotomy from regional veins at the time of the procedure. The patient's age, gender, clinical class, duration of symptoms, presence of superficial truncal reflux by duplex ultrasound, and treatment modality were recorded. Plasma from patients and banked blood samples from healthy volunteers (HVs) were subjected to Luminex (EMD Millipore, Billerica, Mass) to evaluate the expression of an established panel of 20 inflammatory mediators. Mediator concentrations were compared between patients and HVs using Mann-Whitney U tests. Importantly, computational analysis allowed us to compare not only the panel of inflammatory mediators but also the inflammatory networks connecting these mediators to one another. Principal components were analyzed to assess network robustness in each group. RESULTS: CVI venous blood revealed significantly lower levels of monokine induced by γ interferon, soluble interleukin (IL) 2 receptor α chain, IL-4, IL-6, IL-7, tumor necrosis factor α, eotaxin, and granulocyte-macrophage colony-stimulating factor than blood from controls. Inflammatory networks were significantly less complex and less robust in the CVI patients compared with HVs. Based on principal component analysis, responses among HVs were more varied than those of CVI patients. CONCLUSIONS: We demonstrate that patients with CVI have significant differences not only in blood-borne inflammatory mediators but also in the interconnectedness of these mediators with one another and in their principal inflammatory characteristics. Results suggest hypoinflammation in chronic nonhealing changes in CVI. These novel findings, if validated in larger cohorts, may help predict the risk of disease progression or response to therapy in the future and may guide mechanistic studies on tissue responses to CVI.

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.