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.

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.

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.

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.

Trauma in silico: Individual-specific mathematical models and virtual clinical populations.

Trauma-induced critical illness is driven by acute inflammation, and elevated systemic interleukin-6 (IL-6) after trauma is a biomarker of adverse outcomes. We constructed a multicompartment, ordinary differential equation model that represents a virtual trauma patient. Individual-specific variants of this model reproduced both systemic inflammation and outcomes of 33 blunt trauma survivors, from which a cohort of 10,000 virtual trauma patients was generated. Model-predicted length of stay in the intensive care unit, degree of multiple organ dysfunction, and IL-6 area under the curve as a function of injury severity were in concordance with the results from a validation cohort of 147 blunt trauma patients. In a subcohort of 98 trauma patients, those with high-IL-6 single-nucleotide polymorphisms (SNPs) exhibited higher plasma IL-6 levels than those with low IL-6 SNPs, matching model predictions. Although IL-6 could drive mortality in individual virtual patients, simulated outcomes in the overall cohort were independent of the propensity to produce IL-6, a prediction verified in the 98-patient subcohort. In silico randomized clinical trials suggested a small survival benefit of IL-6 inhibition, little benefit of IL-1ß inhibition, and worse survival after tumor necrosis factor-α inhibition. This study demonstrates the limitations of extrapolating from reductionist mechanisms to outcomes in individuals and populations and demonstrates the use of mechanistic simulation in complex diseases.