IL-8 Induces Neutrophil Extracellular Trap Formation in Severe Thermal Injury.

Neutrophil extracellular traps (NETs) have a dual role in the innate immune response to thermal injuries. NETs provide an early line of defence against infection. However, excessive NETosis can mediate the pathogenesis of immunothrombosis, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) in sepsis. Recent studies suggest that high interleukin-8 (IL-8) levels in intensive care unit (ICU) patients significantly contribute to excessive NET generation. This study aimed to determine whether IL-8 also mediates NET generation in patients with severe thermal injuries. IL-8 levels were measured in serum samples from thermally injured patients with ≥15% of the total body surface area (TBSA) and healthy controls (HC). Ex vivo NET generation was also investigated by treating isolated neutrophils with serum from thermal injured patients or normal serum with and without IL-8 and anti-IL-8 antibodies. IL-8 levels were significantly increased compared to HC on days 3 and 5 (p < 0.05) following thermal injury. IL-8 levels were also significantly increased at day 5 in septic versus non-septic patients (p < 0.001). IL-8 levels were also increased in patients who developed sepsis compared to HC at days 3, 5 and 7 (p < 0.001), day 10 (p < 0.05) and days 12 and 14 (p < 0.01). Serum containing either low, medium or high levels of IL-8 was shown to induce ex vivo NETosis in an IL-8-dependent manner. Furthermore, the inhibition of DNase activity in serum increased the NET-inducing activity of IL-8 in vitro by preventing NET degradation. IL-8 is a major contributor to NET formation in severe thermal injury and is increased in patients who develop sepsis. We confirmed that DNase is an important regulator of NET degradation but also a potential confounder within assays that measure serum-induced ex vivo NETosis.

Severe thermal and major traumatic injury results in elevated plasma concentrations of total heme that are associated with poor clinical outcomes and systemic immune suppression.

Background: Traumatic and thermal injuries result in a state of systemic immune suppression, yet the mechanisms that underlie its development are poorly understood. Released from injured muscle and lysed red blood cells, heme is a damage associated molecular pattern with potent immune modulatory properties. Here, we measured plasma concentrations of total heme in over 200 traumatic and thermally-injured patients in order to examine its relationship with clinical outcomes and post-injury immune suppression. Methods: Blood samples were collected from 98 burns (≥15% total body surface area) and 147 traumatically-injured (injury severity score ≥8) patients across the ultra-early (≤1 hour) and acute (4-72 hours) post-injury settings. Pro-inflammatory cytokine production by lipopolysaccharide (LPS) challenged whole blood leukocytes was studied, and plasma concentrations of total heme, and its scavengers haptoglobin, hemopexin and albumin measured, alongside the expression of heme-oxygenase-1 (HO-1) in peripheral blood mononuclear cells (PBMCs). LPS-induced tumour necrosis factor-alpha (TNF-α) production by THP-1 cells and monocytes following in vitro heme treatment was also examined. Results: Burns and traumatic injury resulted in significantly elevated plasma concentrations of heme, which coincided with reduced levels of hemopexin and albumin, and correlated positively with circulating levels of pro and anti-inflammatory cytokines. PBMCs isolated from trauma patients 4-12 and 48-72 hours post-injury exhibited increased HO-1 gene expression. Non-survivors of burn injury and patients who developed sepsis, presented on day 1 with significantly elevated heme levels, with a difference of 6.5 µM in heme concentrations corresponding to a relative 52% increase in the odds of post-burn mortality. On day 1 post-burn, heme levels were negatively associated with ex vivo LPS-induced TNF-α and interleukin-6 production by whole blood leukocytes. THP-1 cells and monocytes pre-treated with heme exhibited significantly reduced TNF-α production following LPS stimulation. This impairment was associated with decreased gene transcription, reduced activation of extracellular signal-regulated kinase 1/2 and an impaired glycolytic response. Conclusions: Major injury results in elevated plasma concentrations of total heme that may contribute to the development of endotoxin tolerance and increase the risk of poor clinical outcomes. Restoration of the heme scavenging system could be a therapeutic approach by which to improve immune function post-injury.

Insights from CTTACC: immune system reset by cellular therapies for chronic illness after trauma, infection, and burn.

BACKGROUND AIMS: In this paper, we present a review of several selected talks presented at the CTTACC conference (Cellular Therapies in Trauma and Critical Care) held in Scottsdale, AZ in May 2023. This conference review highlights the potential for cellular therapies to "reset" the dysregulated immune response and restore physiologic functions to normal. Improvements in medical care systems and technology have increasingly saved lives after major traumatic events. However, many of these patients have complicated post-traumatic sequelae, ranging from short-term multi-organ failure to chronic critical illness. METHODS/RESULTS: Patients with chronic critical illness have been found to have dysregulated immune responses. These abnormal and harmful immune responses persist for years after the initial insult and can potentially be mitigated by treatment with cellular therapies. CONCLUSIONS: The sessions emphasized the need for more research and clinical trials with cellular therapies for the treatment of a multitude of chronic illnesses: post-trauma, radiation injury, COVID-19, burns, traumatic brain injury (TBI) and other chronic infections.

Inflammatory proteins and neutrophil extracellular traps increase in burn blister fluid 24h after burn.

Burn wound blister fluid is a valuable matrix for understanding the biological pathways associated with burn injury. In this study, 152 blister fluid samples collected from paediatric burn wounds at three different hospitals were analysed using mass spectrometry proteomic techniques. The protein abundance profile at different days after burn indicated more proteins were associated with cellular damage/repair in the first 24 h, whereas after this point more proteins were associated with antimicrobial defence. The inflammatory proteins persisted at a high level in the blister fluid for more than 7 days. This may indicate that removal of burn blisters prior to two days after burn is optimal to prevent excessive or prolonged inflammation in the wound environment. Additionally, many proteins associated with the neutrophil extracellular trap (NET) pathway were increased after burn, further implicating NETs in the post-burn inflammatory response. NET inhibitors may therefore be a potential treatment to reduce post-burn inflammation and coagulation pathology and enhance burn wound healing outcomes.

Eschar dissolution and the immunoregulator effect of keratinase on burn wounds.

At present, enzyme debridement preparation has shown a good curative effect on eschar removal of burn wounds. Keratinase has shown great potential in enzymatic debridement because of its good fibrin-degrading ability. In this study, the debridement of keratinase was examined by using a third degree burn wound model in rats. We observed the wound, and keratinase shortened the time of eschar dissolution after debridement. Histopathology and immunofluorescence staining showed that the eschar in the keratinase group became thinner, inflammatory cell infiltration in the wound increased, the fluorescence intensity of the macrophage surface marker CD68 increased, and the CD163/CD86 ratio increased. In bone marrow-derived macrophages (BMDMs), there was no significant difference in the activity of CCK-8 in cells in the keratinase group compared with the control group. The fluorescence intensity of the keratinase group was higher than that of the control group. At 12 h, the cell scratches were obviously closed. The number of migrated Transwell cells increased. Flow cytometry and immunofluorescence analysis showed increased expression of CD206 and Arg-1 and decreased expression of CD86 and iNOS. The gene expression of the Arg-1, iNOS and IL-10 was increased, as shown by qPCR. The secretion of IL-10 was increased and TNF-α was decreased, as shown by ELISA. We concluded that keratinase dissolution of eschar not only has a hydrolytic effect on eschar but may also affect immune regulation to enhance the migration and phagocytosis of macrophages, promote the polarization of macrophages, and further enhance the effect of eschar dissolution. Therefore, keratinase may have good prospects for the debridement of burn wounds.

Extracorporeal Blood Purification in Burns: For Whom, Why, and How?

Patients with serious thermal burn injuries require immediate and specialized care in order to minimize morbidity and mortality. Optimal fluid resuscitation, nutritional support, pulmonary care, burn wound care, and infection control practices represent key aspects of patient care in burn centers. When severely burned, the patient usually presents a systemic inflammatory response syndrome, soon balanced by a counter anti-inflammatory response syndrome. These may lead to immune dysregulation/exhaustion favoring infectious complications that dramatically impair the prognosis of burn patients. This narrative review provides an overview of the main concepts, current understanding, and potential applications of extracorporeal blood purification techniques for burn patient management. Current understanding of burn patients' immune responses is reported. Hypotheses and data on the potential value of immunoregulation are reviewed. Finally, how extracorporeal blood purification may be of interest in this specific population is discussed.

Severity of thermal burn injury is associated with systemic neutrophil activation.

Burn injuries elicit a unique and dynamic stress response which can lead to burn injury progression. Though neutrophils represent crucial players in the burn-induced immunological events, the dynamic secretion pattern and systemic levels of neutrophil-derived factors have not been investigated in detail so far. Serum levels of neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and complement factor C3a were quantified in burn victims over 4 weeks post injury. Furthermore, the potential association with mortality, degree of burn injury, and inhalation trauma was evaluated. In addition, leukocyte, platelet, neutrophil, and lymphocyte counts were assessed. Lastly, we analyzed the association of neutrophil-derived factors with clinical severity scoring systems. Serum levels of NE, MPO, CitH3, and C3a were remarkably elevated in burn victims compared to healthy controls. Leukocyte and neutrophil counts were significantly increased on admission day and day 1, while relative lymphocytes were decreased in the first 7 days post burn trauma. Though neutrophil-derived factors did not predict mortality, patients suffering from 3rd degree burn injuries displayed increased CitH3 and NE levels. Accordingly, CitH3 and NE were elevated in cases with higher abbreviated burn severity indices (ABSI). Taken together, our data suggest a role for neutrophil activation and NETosis in burn injuries and burn injury progression. Targeting exacerbated neutrophil activation might represent a new therapeutic option for severe cases of burn injury.

Recommendations for Influenza Vaccination in Burns Patients Based on a Systematic Review of the Evidence.

Severe burn injury is a serious systemic insult that can lead to life-threatening secondary infections. Immunosuppression, inhalation injury, and prolonged length of hospital stay are factors that predispose patients to severe respiratory tract infections. Furthermore, evidence shows that burns can put one at risk of infection long after the original injury. Currently in the United Kingdom, the annual National Flu Immunisation programme outlines guidance for groups who are deemed high risk and, therefore, eligible for the influenza vaccine. At present, no guidance exists for the administration of the influenza vaccine in burn-injured patients, despite knowledge of immunosuppression. The aim of this literature review is to examine the evidence for associations between burn injury and influenza and, where available, evaluate efficacy of influenza vaccines in this cohort. In addition, literature was searched for the effectiveness of the influenza vaccine in patients 65 years and above and in patients admitted to the intensive care unit (ICU), two domains common to patients with severe burns. Three papers were found to suggest increased susceptibility to influenza following burn injury; however, no papers studying the effectiveness of the influenza vaccine in this group were found. Several studies demonstrated improved outcomes in patients over 65 years and patients admitted to ICU. Following the evaluation of the evidence, this review advocates for the consideration of hospitalized burn patients for the influenza vaccine. We suggest the avoidance of vaccine administration in the acute burn phase. Further prospective clinical trials would be required to validate these findings.

Plasma extracellular vesicles released after severe burn injury modulate macrophage phenotype and function.

Extracellular vesicles (EVs) have emerged as key regulators of immune function across multiple diseases. Severe burn injury is a devastating trauma with significant immune dysfunction that results in an ∼12% mortality rate due to sepsis-induced organ failure, pneumonia, and other infections. Severe burn causes a biphasic immune response: an early (0-72 h) hyper-inflammatory state, with release of damage-associated molecular pattern molecules, such as high-mobility group protein 1 (HMGB1), and proinflammatory cytokines (e.g., IL-1ß), followed by an immunosuppressive state (1-2+ wk post injury), associated with increased susceptibility to life-threatening infections. We have reported that early after severe burn injury HMGB1 and IL-1ß are enriched in plasma EVs. Here we tested the impact of EVs isolated after burn injury on phenotypic and functional consequences in vivo and in vitro using adoptive transfers of EV. EVs isolated early from mice that underwent a 20% total body surface area burn injury (burn EVs) caused similar hallmark cytokine responses in naïve mice to those seen in burned mice. Burn EVs transferred to RAW264.7 macrophages caused similar functional (i.e., cytokine secretion) and immune gene expression changes seen with their associated phase of post-burn immune dysfunction. Burn EVs isolated early (24 h) induced MCP-1, IL-12p70, and IFNγ, whereas EVs isolated later blunted RAW proinflammatory responses to bacterial endotoxin (LPS). We also describe significantly increased HMGB1 cargo in burn EVs purified days 1 to 7 after injury. Thus, burn EVs cause immune outcomes in naïve mice and macrophages similar to findings after severe burn injury, suggesting EVs promote post-burn immune dysfunction.

Ethanol Intoxication and Burn Injury Increases Intestinal Regulatory T Cell Population and Regulatory T Cell Suppressive Capability.

ABSTRACT: Traumatic injuries, such as burn, are often complicated by ethanol intoxication at the time of injury. This leads to a myriad of complications and post-burn pathologies exacerbated by aberrant immune responses. Recent findings suggest that immune cell dysfunction in the gastrointestinal system is particularly important in deleterious outcomes associated with burn injuries. In particular, intoxication at the time of burn injury leads to compromised intestinal T cell responses, which can diminish intestinal immunity and promote bacterial translocation, allowing for increased secondary infections in the injured host and associated sequelae, such as multiple organ failure and sepsis. Regulatory T cells (Treg) have been identified as important mediators of suppressing effector T cell function. Therefore, the goal of this study was to assess the effects of ethanol intoxication and burn injury on Treg populations in small intestinal immune organs. We also evaluated the suppressive capability of Tregs isolated from injured animals. Male C57BL/6 mice were gavaged with 2.9 g/kg ethanol before receiving a ∼12.5% total body surface area scald burn. One day after injury, we identified a significant increase in Tregs number in small intestine Peyer's patches (∼×1.5) and lamina propria (∼×2). Tregs-producing cytokine IL-10 were also increased in both tissues. Finally, Tregs isolated from ethanol and burn-injured mice were able to suppress proliferation of effector T cells to a greater degree than sham vehicle Tregs. This was accompanied by increased levels of IL-10 and decreased levels of pro-proliferative cytokine IL-2 in cultures containing ethanol + burn Tregs compared with sham Tregs. These findings suggest that Treg populations are increased in intestinal tissues 1 day following ethanol intoxication and burn injury. Tregs isolated from ethanol and burn-injured animals also exhibit a greater suppression of effector T cell proliferation, which may contribute to altered T cell responses following injury.

Pharmacological activation of SIRT1 by metformin prevented trauma-induced heterotopic ossification through inhibiting macrophage mediated inflammation.

Trauma-induced heterotopic ossification (HO) is the aberrant extra-skeletal bone formation that severely incapacitates patient's daily life. Inflammation is the first stage of this progression, becoming an appealing target of early therapeutic intervention. Metformin, a widely used antidiabetic drug, also poses the therapeutic potential to modulate various inflammatory-related diseases. Therefore, this study aimed to investigate the preventive effect of metformin on trauma-induced HO progression, and unveil the underlying molecular mechanisms. A murine burn/tenotomy model was established to mimic trauma-induced HO in vivo. The anti-inflammation and anti-ossification effects of metformin were evaluated by histological staining and micro-CT. The inhibitory effects of metformin on macrophages activation in vitro were examined by ELISA and qRT-PCR. The underlying molecular mechanisms were further explored by immunofluorescence staining and western-blotting in vivo. Increased macrophages infiltration and inflammatory responses were found at early stage during HO progression. However, metformin dose-dependently attenuated the macrophage-mediated inflammatory responses both in vivo and vitro, which might account for the inhibitory effect of metformin on chondrogenesis and HO formation after trauma. Furthermore, elevated SIRT1 expression and decreased NF-κB p65 acetylation were found in the beneficial effects of metformin. Moreover, similar preventive effects were also found in SRT1720 HCI, a specific SIRT1 activator, while were remarkably reversed after the administration of EX527 (a specific SIRT1 inhibitor) with metformin. Taken together, our results provide a novel evidence that metformin can effectively attenuate trauma-induced HO by mitigating macrophage inflammatory responses through inhibiting NF-κB signaling via SIRT1-dependent mechanisms, which favors future therapeutic investigations for trauma-related disease.

TPPU treatment of burned mice dampens inflammation and generation of bioactive DHET which impairs neutrophil function.

Oxylipins modulate the behavior of immune cells in inflammation. Soluble epoxide hydrolase (sEH) converts anti-inflammatory epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET). An sEH-inhibitor, TPPU, has been demonstrated to ameliorate lipopolysaccharide (LPS)- and sepsis-induced inflammation via EETs. The immunomodulatory role of DHET is not well characterized. We hypothesized that TPPU dampens inflammation and that sEH-derived DHET alters neutrophil functionality in burn induced inflammation. Outbred mice were treated with vehicle, TPPU or 14,15-DHET and immediately subjected to either sham or dorsal scald 28% total body surface area burn injury. After 6 and 24 h, interleukin 6 (IL-6) serum levels and neutrophil activation were analyzed. For in vitro analyses, bone marrow derived neutrophil functionality and mRNA expression were examined. In vivo, 14,15-DHET and IL-6 serum concentrations were decreased after burn injury with TPPU administration. In vitro, 14,15-DHET impaired neutrophil chemotaxis, acidification, CXCR1/CXCR2 expression and reactive oxygen species (ROS) production, the latter independent from p38MAPK and PI3K signaling. We conclude that TPPU administration decreases DHET post-burn. Furthermore, DHET downregulates key neutrophil immune functions and mRNA expression. Altogether, these data reveal that TPPU not only increases anti-inflammatory and inflammation resolving EET levels, but also prevents potential impairment of neutrophils by DHET in trauma.

The Role of DAMPS in Burns and Hemorrhagic Shock Immune Response: Pathophysiology and Clinical Issues. Review.

Severe or major burns induce a pathophysiological, immune, and inflammatory response that can persist for a long time and affect morbidity and mortality. Severe burns are followed by a "hypermetabolic response", an inflammatory process that can be extensive and become uncontrolled, leading to a generalized catabolic state and delayed healing. Catabolism causes the upregulation of inflammatory cells and innate immune markers in various organs, which may lead to multiorgan failure and death. Burns activate immune cells and cytokine production regulated by damage-associated molecular patterns (DAMPs). Trauma has similar injury-related immune responses, whereby DAMPs are massively released in musculoskeletal injuries and elicit widespread systemic inflammation. Hemorrhagic shock is the main cause of death in trauma. It is hypovolemic, and the consequence of volume loss and the speed of blood loss manifest immediately after injury. In burns, the shock becomes evident within the first 24 h and is hypovolemic-distributive due to the severely compromised regulation of tissue perfusion and oxygen delivery caused by capillary leakage, whereby fluids shift from the intravascular to the interstitial space. In this review, we compare the pathophysiological responses to burns and trauma including their associated clinical patterns.

Altered Genes and Biological Functions in Response to Severe Burns.

Severe burns are acute wounds caused by local heat exposure, resulting in life-threatening systemic effects and poor survival. However, the specific molecular mechanisms remain unclear. First, we downloaded gene expression data related to severe burns from the GEO database (GSE19743, GSE37069, and GSE77791). Then, a gene expression analysis was performed to identify differentially expressed genes (DEGs) and construct protein-protein interaction (PPI) network. The molecular mechanism was identified by enrichment analysis and Gene Set Enrichment Analysis. In addition, STEM software was used to screen for genes persistently expressed during response to severe burns, and receiver operating characteristic (ROC) curve was used to identify key DEGs. A total of 2631 upregulated and 3451 downregulated DEGs were identified. PPI network analysis clustered these DEGs into 13 modules. Importantly, module genes mostly related with immune responses and metabolism. In addition, we identified genes persistently altered during the response to severe burns corresponding to survival and death status. Among the genes with high area under the ROC curve in the PPI network gene, CCL5 and LCK were identified as key DEGs, which may affect the prognosis of burn patients. Gene set variation analysis showed that the immune response was inhibited and several types of immune cells were decreased, while the metabolic response was enhanced. The results showed that persistent gene expression changes occur in response to severe burns, which may underlie chronic alterations in physiological pathways. Identifying the key altered genes may reveal potential therapeutic targets for mitigating the effects of severe burns.

Anti-flagellin IgY antibodies protect against Pseudomonas aeruginosa infection in both acute pneumonia and burn wound murine models in a non-type-specific mode.

Pseudomonas aeruginosa (PA) is one of the most dominant causes of nosocomial infections in burn patients. Increasing emergence of antibiotic-resistant strains highlights the need for novel antimicrobial agents. Flagellin, the main component protein of flagellum, is determined as the major antigen interacting with anti-P. aeruginosa IgY antibodies. The current study was aimed to evaluate the antibacterial potency of IgY antibodies raised against recombinant type A, and B flagellins. The immunogenicity and specificity of IgY antibodies were confirmed through indirect ELISA and western blot analysis, respectively. Anti-flagellin IgYs reduced the motility, biofilm formation and invasion potency of both strains. The cell surface hydrophobicity (CSH) of bacteria was increased upon IgY treatment, and in vitro opsonophagocytosis assay confirmed the high protective potency of specific antibodies via polymorphonuclear leukocyte (PMN)-augmented bacterial cell killing. The protective efficacy of IgYs was also studied in both acute pneumonia and burn wound murine models. Anti-flagellin B-IgY induced 100 % and 40 % protection against laboratory, and hospital strains in burn wound model, respectively. Protection in acute pneumonia against all strains was 100 %. Anti-flagellin A-IgY failed to protect mice in burn wound model, but provided 100 % protection against all strains in acute pneumonia challenge. In vitro, ex vivo and in vivo experiments confirmed the dose-dependent and non-type specific essence of anti-flagellin IgY antibodies, providing the benefit of covering all strain types in a dose dependent manner. Our findings provide evidence that anti-flagellin IgY antibodies qualify as novel economical therapeutic option against PA infection.

A Nonlethal Murine Flame Burn Model Leads to a Transient Reduction in Host Defenses and Enhanced Susceptibility to Lethal Pseudomonas aeruginosa Infection.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.

Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult.

Heterotopic ossification (HO) is one of the most intractable disorders following musculoskeletal injury and is characterized by the ectopic presence of bone tissue in the soft tissue leading to severe loss of function in the extremities. Recent studies have indicated that immune cell infiltration and inflammation are involved in aberrant bone formation. In this study, we found increased monocyte/macrophage and mast cell accumulation during early HO progression. Macrophage depletion by clodronate liposomes and mast cell stabilization by cromolyn sodium significantly impeded HO formation. Therefore, we proposed that the dietary phytochemical quercetin could also suppress immune cell recruitment and related inflammatory responses to prevent HO. As expected, quercetin inhibited the monocyte-to-macrophage transition, macrophage polarization, and mast cell activation in vitro in a dose-dependent manner. Using a murine burn/tenotomy model, we also demonstrated that quercetin attenuated inflammatory responses and HO in vivo. Furthermore, elevated SIRT1 and decreased acetylated NFκB p65 expression were responsible for the mechanism of quercetin, and the beneficial effects of quercetin were reversed by the SIRT1 antagonist EX527 and mimicked by the SIRT agonist SRT1720. The findings in this study suggest that targeting monocyte/macrophage and mast cell activities may represent an attractive approach for therapeutic intervention of HO and that quercetin may serve as a promising therapeutic candidate for the treatment of trauma-induced HO by modulating SIRT1/NFκB signaling.

STIM1 Regulates Endothelial Calcium Overload and Cytokine Upregulation During Sepsis.

BACKGROUND: Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) is now recognized as the main mechanism of the majority of nonexcitable cell calcium influx. Calcium overload is a primary mechanism of endothelial cell injury during systemic inflammatory response and sepsis. Whether STIM1-mediated SOCE plays a role in calcium overload in vascular endothelial cell injury remains unclear. MATERIALS AND METHODS: To explore the role of STIM1-gated SOCE in vascular endothelial cell calcium overload and inflammation, we established a human septic serum or lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell (HUVEC) experimental system and derived ribonucleic acid interference (RNAi)-mediated STIM1, ORAI1 (orai gene [HGNC: 25896 Entrez Gene: 84876] coding protein, ORAI Calcium Release-Activated Calcium Modulator 1), and transient receptor potential channel 1 (TRPC1) (core components of store-operated Ca2+[SOC]) downregulated HUVECs, as well as STIM1 overinduced HUVECs. RESULTS: Our results show that sepsis serum or LPS stimulation increased STIM1 in HUVECs and increased all cytokines except for VEGF and the inflammatory mediators tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1 in a time-dependent manner. RNAi-mediated knockdown of STIM1 significantly inhibited serum or LPS-induced inflammatory cytokine expression, and STIM1 overexpression in HUVECs promoted LPS-mediated induction of these cytokines. Meanwhile, similar to the blocking effect of the specific SOC inhibitors Gd3+ and La3+ on LPS-induced calcium influx, RNAi-mediated depletion of STIM1 or the SOC proteins TRPC1 and ORAI1 could significantly inhibit serum or LPS-induced extracellular calcium influx, as well as the expression of the inflammatory cytokines tumor necrosis factor, intercellular cell adhesion molecule-1, and endothelin-1. Simultaneous downregulation of the SOCE core units TRPC1 and ORAI1 inhibited LPS-induced calcium influx and cytokine expression, which could not be restored by inducing STIM1. Forced expression of nuclear factor-κB (NF-κB) in HUVECs significantly induced STIM1 expression, whereas RNAi-mediated depletion of NF-κB significantly inhibited STIM1 mRNA levels and significantly reduced the thapsigargin-mediated SOCE calcium influx, which was similar to results with the NF-κB inhibitor wogonin. CONCLUSIONS: Septic serum stimulates the expression of STIM1, cytokines, and inflammatory mediators in HUVECs. STIM1-mediated SOCE is required for Ca2+ influx induced by LPS or septic serum and contributes cytokines and inflammatory mediators in septic serum-stimulated HUVECs. In addition, STIM1-mediated SOCE on Ca2+ influx by septic serum or LPS involves NF-κB signaling.

Post-injury immunosuppression and secondary infections are caused by an AIM2 inflammasome-driven signaling cascade.

Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1ß (IL-1ß) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.