ALTERED PROFILES OF EXTRACELLULAR MITOCHONDRIAL DNA IN IMMUNOPARALYZED PEDIATRIC PATIENTS AFTER THERMAL INJURY.

ABSTRACT: Background: Thermal injury is a major cause of morbidity and mortality in the pediatric population worldwide with secondary infection being the most common acute complication. Suppression of innate and adaptive immune function is predictive of infection in pediatric burn patients, but little is known about the mechanisms causing these effects. Circulating mitochondrial DNA (mtDNA), which induces a proinflammatory signal, has been described in multiple disease states but has not been studied in pediatric burn injuries. This study examined the quantity of circulating mtDNA and mtDNA mutations in immunocompetent (IC) and immunoparalyzed (IP) pediatric burn patients. Methods: Circulating DNA was isolated from plasma of pediatric burn patients treated at Nationwide Children's Hospital Burn Center at early (1-3 days) and late (4-7 days) time points postinjury. These patients were categorized as IP or IC based on previously established immune function testing and secondary infection. Three mitochondrial genes, D loop, ND1, and ND4, were quantified by multiplexed qPCR to assess both mtDNA quantity and mutation load. Results: At the early time point, there were no differences in plasma mtDNA quantity; however, IC patients had a progressive increase in mtDNA over time when compared with IP patients (change in ND1 copy number over time 3,880 vs. 87 copies/day, P = 0.0004). Conversely, the IP group had an increase in mtDNA mutation burden over time. Conclusion: IC patients experienced a significant increase in circulating mtDNA quantity over time, demonstrating an association between increased mtDNA release and proinflammatory phenotype in the burn patients. IP patients had significant increases in mtDNA mutation load likely representative of degree of oxidative damage. Together, these data provide further insight into the inflammatory and immunological mechanisms after pediatric thermal injury.

DEMOGRAPHICS TO DEFINE PEDIATRIC BURN PATIENTS AT RISK OF ADVERSE OUTCOMES.

ABSTRACT: Background: There is currently no standard definition of a severe burn in the pediatric patient population to identify those at higher risk of infectious complications. Our aim was to correlate total burn surface area (TBSA), burn depth, and type of burn injury to nosocomial infection rates and systemic immune system responses to better define risk factors associated with adverse outcomes. Methods: A prospective observational study at a single-center, quaternary-care, American Burn Association-verified pediatric burn center was conducted from 2016 to 2021. Blood was collected within 72 h of injury from 103 pediatric patients. Whole blood was incubated with lipopolysaccharide or phytohemagglutinin stimulation reagent to measure innate and adaptive immune response, respectively. Flow cytometry was performed on whole blood samples to measure both innate and adaptive immune cells. Unstimulated plasma was also extracted, and IL-6 and IL-10 as well as soluble proteins B- and T-lymphocyte attenuator, CD27, and T-cell immunoglobulin mucin 3 were quantified. Results: There was a significant increased risk for nosocomial infection in pediatric patients with TBSA burns of ≥20%, full-thickness burn injuries ≥5%, or flame burn injuries. There was an overall decrease in both innate and adaptive immune function in patients with TBSA burns ≥20% or full-thickness burn injuries ≥5%. Both burn injury characteristics were also associated with a significant increase in unstimulated IL-6 and IL-10 and soluble immunoregulatory checkpoint proteins. We observed a significant decrease in soluble B- and T-lymphocyte attenuator for those with a flame injury, but there were no other differences between flame injury and scald/contact burns in terms of innate and adaptive immune function. Conclusion: Burns with ≥20% TBSA or ≥5% full thickness in pediatric patients are associated with systemic immune dysfunction and increased risk of nosocomial infections.

Early detection of soluble CD27, BTLA, and TIM-3 predicts the development of nosocomial infection in pediatric burn patients.

Thermal injury induces concurrent inflammatory and immune dysfunction, which is associated with adverse clinical outcomes. However, these effects in the pediatric population are less studied and there is no standard method to identify those at risk for developing infections. Our goal was to better understand immune dysfunction and identify soluble protein markers following pediatric thermal injury. Further we wanted to determine which early inflammatory, soluble, or immune function markers are most predictive of the development of nosocomial infections (NI) after burn injury. We performed a prospective observational study at a single American Burn Association-verified Pediatric Burn Center. A total of 94 pediatric burn subjects were enrolled and twenty-three of those subjects developed a NI with a median time to diagnosis of 8 days. Whole blood samples, collected within the first 72 hours after injury, were used to compare various markers of inflammation, immune function, and soluble proteins between those who recovered without developing an infection and those who developed a NI after burn injury. Within the first three days of burn injury, innate and adaptive immune function markers (ex vivo lipopolysaccharide-induced tumor necrosis factor alpha production capacity, and ex vivo phytohemagglutinin-induced interleukin-10 production capacity, respectively) were decreased for those subjects who developed a subsequent NI. Further analysis of soluble protein targets associated with these pathways displayed significant increases in soluble CD27, BTLA, and TIM-3 for those who developed a NI. Our findings indicate that suppression of both the innate and adaptive immune function occurs concurrently within the first 72 hours following pediatric thermal injury. At the same time, subjects who developed NI have increased soluble protein biomarkers. Soluble CD27, BTLA, and TIM-3 were highly predictive of the development of subsequent infectious complications. This study identifies early soluble protein makers that are predictive of infection in pediatric burn subjects. These findings should inform future immunomodulatory therapeutic studies.

The role of macrophages in thermal injury.

Macrophages, first discovered for their phagocytic ability, are a complicated and heterogeneous cell type. The unique properties of macrophages allow them to perform a vast array of functions, including phagocytosis, cytokine production, antigen presentation, and wound healing. Some macrophage populations are derived from monocytes and are induced into specific phenotypes by the local tissue microenvironment, while other macrophages form during early embryonic development. The exposure of the host to local pathogens and/or traumatic injury alters the tissue microenvironment and, in turn, influences changes in macrophage phenotype and function. Perhaps the most significant change in the local tissue microenvironment and subsequent macrophage phenotype occurs after thermal injury, which causes localized tissue damage and a massive systemic inflammatory response. However, few studies have explored the influence of burn injury on the host macrophages and macrophage function in burn wounds. Furthermore, the literature is scant regarding the impact macrophage function has on outcomes in thermal injury. This review will focus on the current knowledge of macrophage function in burn wounds and the phenotypic changes in macrophages during thermal injury while identifying knowledge gaps.

Corticosterone and chlorpyrifos oxon exposure elicits spatiotemporal MAPK phosphoprotein signaling in a mouse brain.

Chlorpyrifos (CPF) is one of the most widely-used pesticides globally for agricultural purposes. Certain occupations (e.g., farmers, military) are at an increased risk for high-dose exposure to CPF, which can lead to seizures and irreversible brain injury. Workers with the highest risk of exposure typically experience increased circulating cortisol levels, which is related to physiological stress. To better represent this exposure scenario, a mouse model utilized exogenous administration of corticosterone (CORT; high physiologic stress mimic) in combination with chlorpyrifos oxon (CPO; oxon metabolite of CPF); this combination increases neuroinflammation post-exposure. In the present study adult male C57BL/6J mice were given CORT (200 µg/mL) in drinking water for seven days followed by a single intraperitoneal injection of CPO (8.0 mg/kg) on day eight, and euthanized 0.5, 2, and 24 h post-injection. Ten post-translationally modified proteins were measured in the frontal cortex and striatum to evaluate brain region-specific effects. The spatiotemporal response to CORT + CPO sequentially activated phosphoproteins (p-ERK1/2, p-MEK1/2, p-JNK) involved in mitogen-activated protein kinase (MAPK) signaling. Observed p-ZAP70 responses further integrated MAPK signaling and provided a spatiotemporal connection between protein phosphorylation and neuroinflammation. This study provides insight into the spatiotemporal cellular signaling cascade following CORT + CPO exposure that represent these vulnerable populations.

Differential phosphoprotein signaling in the cortex in mouse models of Gulf War Illness using corticosterone and acetylcholinesterase inhibitors.

AIMS: Veterans from the 1990-91 Gulf War were exposed to acetylcholinesterase inhibitors (AChEIs), and, following service, an estimated one-third began suffering from a medically unexplained, multi-symptom illness termed Gulf War Illness (GWI). Previous research has developed validated rodent models that include exposure to exogenous corticosterone (CORT) and AChEIs to simulate high stress and chemical exposures encountered in theater. This combination of exposures in mice resulted in a marked increase in neuroinflammation, which is a common symptom of veterans suffering from GWI. To further elucidate the mechanisms associated with these mouse models of GWI, an investigation into intracellular responses in the cortex were performed to characterize the early cellular signaling changes associated with this exposure-initiated neuroinflammation. MAIN METHODS: Adult male C57BL/6J mice were exposed to CORT in the drinking water (200 µg/mL) for 7 days followed by a single intraperitoneal injection of diisopropyl fluorophosphate (DFP; 4.0 mg/kg) or chlorpyrifos oxon (CPO; 8.0 mg/kg), on day 8 and euthanized 0.5, 2, and 24 h post-injection. Eleven post-translationally modified protein targets were measured using a multiplexed ELISA. KEY FINDINGS: Phosphoprotein responses were found to be exposure specific following AChEI insult, with and without CORT. Specifically, CORT + CPO exposure was found to sequentially activate several phosphoproteins involved in mitogen activated protein kinase signaling (p-MEK1/2, p-ERK1/2, and p-JNK). DFP alone similarly increased proteins in this pathway (p-RPS6, and p-JNK), but the addition of CORT ameliorated these affects. SIGNIFICANCE: The results of this study provide insight into differentially activated pathways depending on AChEI in these GWI models.

Tissue-level cytokines in a rodent model of chronic implant-associated infection.

Systemic cytokine concentrations have been extensively studied in implant-associated infections, providing sensitive diagnostic markers. However, less is known about the relationships of tissue-level cytokines surrounding the joint. The aim of this study was to define the cytokine profiles of tissues to investigate the use of these cytokines as markers of debridement in chronic joint infection. Using a rodent model, muscle samples were obtained from rats following Kirschner wire implantation and infection with Staphylococcus aureus to determine if: (1) differences exist in cytokine concentrations with proximity to infection, and (2) localized infection-specific markers can be identified on a tissue level to potentially serve as debridement markers in the future. Samples were collected from 4 distinct locations, and the concentrations of interleukin(IL)-1α, IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, granulocyte-macrophage colony-stimulating factor, interferon-γ, and tumor necrosis factor-α were quantified in each sample, relative to the amount of tissue. Cytokine concentrations differed with proximity to the joint when implant or infection was present, and tissues at the operative knee joint showed the highest levels of most cytokines. Additionally, IL-1ß, IL-4, and IL-6 showed promise, beyond diagnostics, as tissue-level indicators of infection response. Ultimately, this study illustrated that tissue-level evaluation provided insight into infection-specific response, and these markers may be useful for guiding the debridement of implant-associated infections.

Localized cytokine responses to total knee arthroplasty and total knee revision complications.

BACKGROUND: The study of localized immune-related factors has proven beneficial for a variety of conditions, and one area of interest in the field of orthopaedics is the impact of implants and localized infections on immune response. Several cytokines have shown increased systemic concentrations (in serum/plasma) in response to implants and infection, but tissue-level cytokines have not been investigated as thoroughly. METHODS: This exploratory study investigated tissue-level cytokines in a cohort of patients (N = 17) in response to total knee arthroplasty and total knee revision to better understand the immune response to implants and localized infection (e.g., prosthetic joint infection). The overall goal of this study was to provide insight into the localized cytokine response of tissues and identify tissue-level markers specific to inflammation caused by implants vs. inflammation caused by infection. Tissues were collected across several anatomical locations and assayed with a panel of 20 human inflammatory cytokines to understand spatial differences in cytokine levels. RESULTS: In this study, six cytokines were elevated in implanted joints, as compared to native joints: IL-10, IL-12p70, IL-13, IL-17A, IL-4, and TNF-α (p < 0.05). Seven cytokines showed infection-dependent increases in localized tissues: IL-1α, IL-1ß, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1ß (p < 0.05). CONCLUSIONS: This study demonstrated that differences exist in tissue-level cytokines in response to presence of implant, and some cytokines were specifically elevated for infection; these responses may be informative of overall tissue health. These results highlight the utility of investigating localized cytokine concentrations to offer novel insights for total knee arthroplasty and total knee revision procedures, as well as their complications. Ultimately, this information could provide additional, quantitative measurements of tissue to aid clinical decision making and patient treatment options.

Impact of Cytokines and Phosphoproteins in Response to Chronic Joint Infection.

The early cellular response to infection has been investigated extensively, generating valuable information regarding the mediators of acute infection response. Various cytokines have been highlighted for their critical roles, and the actions of these cytokines are related to intracellular phosphorylation changes to promote infection resolution. However, the development of chronic infections has not been thoroughly investigated. While it is known that wound healing processes are disrupted, the interactions of cytokines and phosphoproteins that contribute to this dysregulation are not well understood. To investigate these relationships, this study used a network centrality approach to assess the impact of individual cytokines and phosphoproteins during chronic inflammation and infection. Tissues were taken from patients undergoing total knee arthroplasty (TKA) and total knee revision (TKR) procedures across two tissue depths to understand which proteins are contributing most to the dysregulation observed at the joint. Notably, p-c-Jun, p-CREB, p-BAD, IL-10, IL-12p70, IL-13, and IFN-γ contributed highly to the network of proteins involved in aseptic inflammation caused by implants. Similarly, p-PTEN, IL-4, IL-10, IL-13, IFN-γ, and TNF-α appear to be central to signaling disruptions observed in septic joints. Ultimately, the network centrality approach provided insight into the altered tissue responses observed in chronic inflammation and infection.

Bioenergetic Analyses of In Vitro and In Vivo Samples to Guide Toxicological Endpoints.

Toxicology is a broad field that requires the translation of biochemical responses to xenobiotic exposures into useable information to ensure the safety of the public. Modern techniques are improving rapidly, both quantitatively and qualitatively, to provide the tools necessary to expand available toxicological datasets and refine our ability to translate that data into relevant information via bioinformatics. These new techniques can, and do, impact many of the current critical roles in toxicology, including the environmental, forensic, preclinical/clinical, and regulatory realms. One area of rapid expansion is our understanding of bioenergetics, or the study of the transformation of energy in living organisms, and new mathematical approaches are needed to interpret these large datasets. As bioenergetics are intimately involved in the regulation of how and when a cell responds to xenobiotics, monitoring these changes (i.e., metabolic fluctuations) in cells/tissues post-exposure provides an approach to define the temporal scale of pharmacodynamic responses, which can be used to guide additional toxicological techniques (e.g., "omics"). This chapter will summarize important in vitro assays and in vivo imaging techniques to take real-time measurements. Using this information, our laboratory has utilized bioenergetics to identify significant time points of pharmacodynamic relevance as well as forecast the cell's eventual fate.

Salivary cytokines as a biomarker of social stress in a mock rescue mission.

Using salivary inflammatory markers as a noninvasive biomonitoring technique within natural social contexts has become increasingly important to link social and biological responses. Many studies have associated circulating cytokines to distinct aspects of physical activity and social/emotional behavior; however, they have not been linked to success and failure in a naturalistic setting for military personnel performing tasks. In this study, salivary cytokines were studied in a group of fifteen Air Force Reserve Officers' Training Corps (ROTC; 14 males, 1 female) subjects performing three mock hostage rescue missions, designed to prompt responses associated with baseline, success, and failure. Each subject completed the tasks of the mission individually and again in randomly assigned teams. Participants were outfitted via direct skin contact with comfortable external Zephyr™ sensors to monitor heart rate, breathing rate, and activity while completing each task. Saliva samples were collected before and after the completion of each mission, and cytokine levels were quantified using enzyme-labelled immunoassay (ELISA) beads. These biomarkers were used to describe the body's immune response to success and failure when performing a mock rescue mission individually and in a team. All measured cytokine levels increased following failed missions performed individually, compared to cytokine levels associated with successful missions. When completing the tasks as a team, there were no significant differences in cytokine response between success and failure; however, being in a team stimulated an increased pre-mission cytokine response, suggesting the concept of teamwork and performing with peers for the first time had a more significant impact than the notion of failing. Additionally, none of the cytokines tested for individual missions correlated to physical activity markers (heart rate, breathing rate, activity) measured during performance. These results indicate a potentially new noninvasive method of determining social stress levels under taxing conditions.