Antimicrobial activities of lavandulylated flavonoids in Sophora flavences against methicillin-resistant Staphylococcus aureus via membrane disruption.

INTRODUCTION: The continuous emergence and rapid spread of multidrug-resistant bacteria have accelerated the demand for the discovery of alternative antibiotics. Natural plants contain a variety of antibacterial components, which is an important source for the discovery of antimicrobial agents. OBJECTIVE: To explore the antimicrobial activities and related mechanisms of two lavandulylated flavonoids, sophoraflavanone G and kurarinone in Sophora flavescens against methicillin-resistant Staphylococcus aureus. METHODS: The effects of sophoraflavanone G and kurarinone on methicillin-resistant Staphylococcus aureus were comprehensively investigated by a combination of proteomics and metabolomics studies. Bacterial morphology was observed by scanning electron microscopy. Membrane fluidity, membrane potential, and membrane integrity were determined using the fluorescent probes Laurdan, DiSC3(5), and propidium iodide, respectively. Adenosine triphosphate and reactive oxygen species levels were determined using the adenosine triphosphate kit and reactive oxygen species kit, respectively. The affinity activity of sophoraflavanone G to the cell membrane was determined by isothermal titration calorimetry assays. RESULTS: Sophoraflavanone G and kurarinone showed significant antibacterial activity and anti-multidrug resistance properties. Mechanistic studies mainly showed that they could target the bacterial membrane and cause the destruction of the membrane integrity and biosynthesis. They could inhibit cell wall synthesis, induce hydrolysis and prevent bacteria from synthesizing biofilms. In addition, they can interfere with the energy metabolism of methicillin-resistant Staphylococcus aureus and disrupt the normal physiological activities of the bacteria. In vivo studies have shown that they can significantly improve wound infection and promote wound healing. CONCLUSION: Kurarinone and sophoraflavanone G showed promising antimicrobial properties against methicillin-resistant Staphylococcus aureus, suggesting that they may be potential candidates for the development of new antibiotic agents against multidrug-resistant bacteria.

Wheat Germ-Derived Peptide Alleviates Dextran Sulfate Sodium-Induced Colitis in Mice.

This study explores the protective properties and potential mechanisms of wheat-germ-derived peptide APEPEPAF (APE) against ulcerative colitis. Colitis mice induced by dextran sulfate sodium (DSS) were used as the animal model. The results showed that the APE peptide could alleviate colitis symptoms including weight loss, colon shortening, and histopathological changes. This peptide attenuated the generation of inflammatory cytokines by inhibiting the phosphorylation of protein kinase PKCζ (Thr410) and NF-κB transcriptional activity in DSS-induced mice, suggesting that APE ameliorates colitis inflammation by regulating the PKCζ/NF-κB signaling pathway. APE also preserved the barrier function of the colon by dose-dependently promoting the expression of tight junction proteins (claudin-1, zonula occluded-1, and occludin). In addition, APE significantly decreased the abundance of Bacteroides and increased the abundance of Dubosiella and Lachnospiraceae_UCG-006 to improve the intestinal flora imbalance in DSS-induced colitis mice. Therefore, wheat germ peptide APE can be used as a novel agent and dietary supplement to treat ulcerative colitis..

Cytomegalovirus durably primes neutrophil oxidative burst.

Cytomegalovirus (CMV) is a ubiquitous herpes virus that infects most humans, thereafter persisting lifelong in tissues of the host. It is a known pathogen in immunosuppressed patients, but its impact on immunocompetent hosts remains less understood. Recent data have shown that CMV leaves a significant and long-lasting imprint in host immunity that may confer some protection against subsequent bacterial infection. Such innate immune activation may come at a cost, however, with potential to cause immunopathology. Neutrophils are central to many models of immunopathology, and while acute CMV infection is known to influence neutrophil biology, the impact of chronic CMV infection on neutrophil function remains unreported. Using our murine model of CMV infection and latency, we show that chronic CMV causes persistent enhancement of neutrophil oxidative burst well after resolution of acute infection. Moreover, this in vivo priming of marrow neutrophils is associated with enhanced formyl peptide receptor expression, and ultimately constitutive c-Jun N-terminal kinase phosphorylation and enhanced CD14 expression in/on circulating neutrophils. Finally, we show that neutrophil priming is dependent on viral load, suggesting that naturally infected human hosts will show variability in CMV-related neutrophil priming. Altogether, these findings represent a previously unrecognized and potentially important impact of chronic CMV infection on neutrophil responsiveness in immunocompetent hosts.

DANGER Signals Activate G -Protein Receptor Kinases Suppressing Neutrophil Function and Predisposing to Infection After Tissue Trauma.

OBJECTIVE: Injured tissue predisposes the subject to local and systemic infection. We studied injury-induced immune dysfunction seeking novel means to reverse such predisposition. BACKGROUND: Injury mobilizes primitive "DANGER signals" [danger-associated molecular patterns (DAMPs)] activating innate immunocyte (neutrophils, PMN) signaling and function. Mitochondrial formyl peptides activate G -protein coupled receptors (GPCR) like formyl peptide receptor-1. Mitochondrial DNA and heme activate toll-like receptors (TLR9 and TLR2/4). GPCR kinases (GRKs) can regulate GPCR activation. METHODS: We studied human and mouse PMN signaling elicited by mitochondrial DAMPs (GPCR surface expression; protein phosphorylation, or acetylation; Ca 2+ flux) and antimicrobial functions [cytoskeletal reorganization, chemotaxis (CTX), phagocytosis, bacterial killing] in cellular systems and clinical injury samples. Predicted rescue therapies were assessed in cell systems and mouse injury-dependent pneumonia models. RESULTS: Mitochondrial formyl peptides activate GRK2, internalizing GPCRs and suppressing CTX. Mitochondrial DNA suppresses CTX, phagocytosis, and killing through TLR9 through a novel noncanonical mechanism that lacks GPCR endocytosis. Heme also activates GRK2. GRK2 inhibitors like paroxetine restore functions. GRK2 activation through TLR9 prevented actin reorganization, implicating histone deacetylases (HDACs). Actin polymerization, CTX, bacterial phagocytosis, and killing were also rescued, therefore, by the HDAC inhibitor valproate. Trauma repository PMN showed GRK2 activation and cortactin deacetylation, which varied with severity and was most marked in patients developing infections. Either GRK2 or HDAC inhibition prevented loss of mouse lung bacterial clearance, but only the combination rescued clearance when given postinjury. CONCLUSIONS: Tissue injury-derived DAMPs suppress antimicrobial immunity through canonical GRK2 activation and a novel TLR-activated GRK2-pathway impairing cytoskeletal organization. Simultaneous GRK2/HDAC inhibition rescues susceptibility to infection after tissue injury.

Can Neutrophils Prevent Nosocomial Pneumonia after Serious Injury?

Nosocomial pneumonia is a leading cause of critical illness and mortality among seriously injured trauma patients. However, the link between injury and the development of nosocomial pneumonia is still not well recognized. Our work strongly suggests that mitochondrial damage-associated molecular patterns (mtDAMPs), especially mitochondrial formyl peptides (mtFPs) released by tissue injury, play a significant role in developing nosocomial pneumonia after a serious injury. Polymorphonuclear leukocytes (neutrophils, PMN) migrate toward the injury site by detecting mtFPs through formyl peptide receptor 1 (FPR1) to fight/contain bacterial infection and clean up debris. Activation of FPR1 by mtFPs enables PMN to reach the injury site; however, at the same time it leads to homo- and heterologous desensitization/internalization of chemokine receptors. Thus, PMN are not responsive to secondary infections, including those from bacteria-infected lungs. This may enable a progression of bacterial growth in the lungs and nosocomial pneumonia. We propose that the intratracheal application of exogenously isolated PMN may prevent pneumonia coupled with a serious injury.

Plasma and wound fluids from trauma patients suppress neutrophil extracellular respiratory burst.

BACKGROUND: Trauma increases susceptibility to secondary bacterial infections. The events suppressing antimicrobial immunity are unclear. Polymorphonuclear neutrophils (PMNs) migrate toward bacteria using chemotaxis, trap them in extracellular neutrophil extracellular traps, and kill them using respiratory burst (RB). We hypothesized that plasma and wound fluids from trauma patients alter PMN function. METHODS: Volunteer PMNs were incubated in plasma or wound fluids from trauma patients (days 0 and 1, days 2 and 3), and their functions were compared with PMNs incubated in volunteer plasma. Chemotaxis was assessed in transwells. Luminometry assessed total and intracellular RB responses to receptor-dependent and independent stimulants. Neutrophil extracellular trap formation was assessed using elastase assays. The role of tissue necrosis in creating functionally suppressive systemic PMN environments was assessed using a novel pig model where PMNs were incubated in uninjured pig plasma or plasma from pigs undergoing intraperitoneal instillation of liver slurry. RESULTS: Both plasma and wound fluids from trauma patients markedly suppress total PMN RB. Intracellular RB is unchanged, implicating suppression of extracellular RB. Wound fluids are more suppressive than plasma. Biofluids suppressed RB maximally early after injury and their effects decayed with time. Chemotaxis and neutrophil extracellular trap formation were suppressed by biofluids similarly. Lastly, plasma from pigs undergoing abdominal liver slurry instillation suppressed PMN RB, paralleling suppression by human trauma biofluids. CONCLUSION: Trauma plasma and wound fluids suppress RB and other key PMNs antimicrobial functions. Circulating suppressive signals can be derived from injured or necrotic tissue at wound sites, suggesting a key mechanism by which tissue injuries can put the host at risk for infection.

Role of Mitochondria-Derived Danger Signals Released After Injury in Systemic Inflammation and Sepsis.

Significance: Sepsis is a major public health concern, with high mortality and morbidity, especially among patients undergoing trauma. It is characterized by a systemic inflammatory response syndrome (SIRS) occurring in response to infection. Although classically associated with pathogens, many patients with SIRS do not have infection. The variability of the disease course cannot be fully explained by our current understanding of its pathogenesis. Thus, other factors are likely to play key roles in the development and progression of SIRS/sepsis. Recent Advances: Circulating levels of damage-associated molecular patterns (DAMPs) seem to correlate with SIRS/sepsis morbidity and mortality. Of the known DAMPs, those of mitochondrial (mt) origin have been of particular interest, since their DNA (mtDNA) and formyl peptides (mtFPs) resemble bacterial DNA and peptides, and hence, when released, may be recognized as "danger signals." Critical Issues: mtDAMPs released after tissue injury trigger immune responses similar to those induced by pathogens. Thus, they can result in systemic inflammation and organ damage, similar to that observed in SIRS/sepsis. We will discuss recent findings on the roles of mtDAMPs, particularly regarding the less recognized mtFPs, in the activation of inflammatory responses and development of SIRS/sepsis. Future Directions: There are no established methods to predict the course of SIRS/sepsis, but clinical studies reveal that plasma levels of mtDAMPs may correlate with the outcome of the disease. We propose that non-pathogen-initiated, mtDAMPs-induced SIRS/sepsis events need further studies aimed at early clinical recognition and better treatment of this disease.

Circulating mitochondrial N-formyl peptides contribute to secondary nosocomial infection in patients with septic shock.

Secondary infections typically worsen outcomes of patients recovering from septic shock. Neutrophil [polymorphonuclear leukocytes (PMNs)] migration to secondarily inoculated sites may play a key role in inhibiting progression from local bacterial inoculation to secondary infection. Mitochondrial N-formyl peptide (mtFP) occupancy of formyl peptide receptor-1 (FPR1) has been shown to suppress PMN chemotaxis. Therefore, we studied the association between circulating mtFPs and the development of secondary infection in patients with septic shock. We collected clinical data and plasma samples from patients with septic shock admitted to the intensive care unit for longer than 72 h. Impacts of circulating nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) upon clinical outcomes were analyzed. Next, the role of ND6 in PMN chemotaxis was investigated using isolated human PMNs. Studying plasma samples from 97 patients with septic shock, we found that circulating ND6 levels at admission were independently and highly associated with the development of secondary infection (odds ratio = 30.317, 95% CI: 2.904 to 316.407, P = 0.004) and increased 90-d mortality (odds ratio = 1.572, 95% CI: 1.002 to 2.465, P = 0.049). In ex vivo experiments, ND6 pretreatment suppressed FPR1-mediated PMN chemotactic responses to bacterial peptides in the presence of multiple cytokines and chemokines, despite increased nondirectional PMN movements. Circulating mtFPs appear to contribute to the development of secondary infection and increased mortality in patients with septic shock who survive their early hyperinflammatory phase. The increased susceptibility to secondary infection is probably partly mediated by the suppression of FPR1-mediated PMN chemotaxis to secondary infected sites.

Monocyte exocytosis of mitochondrial danger-associated molecular patterns in sepsis suppresses neutrophil chemotaxis.

BACKGROUND: Trauma and sepsis both increase the risk for secondary infections. Injury mobilizes mitochondrial (MT) danger-associated molecular patterns (mtDAMPs) directly from cellular necrosis. It is unknown, however, whether sepsis can cause active MT release and whether mtDAMPs released by sepsis might affect innate immunity. METHODS: Mitochondrial release from human monocytes (Mo) was studied after LPS stimulation using electron microscopy and using fluorescent video-microscopy of adherent Mo using Mito-Tracker Green (MTG) dye. Release of MTG+ microparticles was studied using flow cytometry after bacterial stimulation by size exclusion chromatography of supernatants with polymerase chain reaction (PCR) for mitochondrial DNA (mtDNA). Human neutrophil (PMN), chemotaxis, and respiratory burst were studied after PMN incubation with mtDNA. RESULTS: LPS caused Mo to release mtDAMPs. Electron microscopy showed microparticles containing MT. mtDNA was present both in microvesicles and exosomes as shown by PCR of the relevant size exclusion chromatography bands. In functional studies, PMN incubation with mtDNA suppressed chemotaxis in a dose-dependent manner, which was reversed by chloroquine, suggesting an endosomal, toll-like receptor-9-dependent mechanism. In contrast, PMN respiratory burst was unaffected by mtDNA. CONCLUSION: In addition to passive release of mtDAMPs by traumatic cellular disruption, inflammatory and infectious stimuli cause active mtDAMP release via microparticles. mtDNA thus released can have effects on PMN that may suppress antimicrobial function. mtDAMP-mediated "feed-forward" mechanisms may modulate immune responses and potentially be generalizable to other forms of inflammation. Where they cause immune dysfunction the effects can be mitigated if the pathways by which the mtDAMPs act are defined. In this case, the endosomal inhibitor chloroquine is benign and well tolerated. Thus, it may warrant study as a prophylactic antiinfective after injury or prior sepsis.

Direct Airway Instillation of Neutrophils Overcomes Chemotactic Deficits Induced by Injury.

BACKGROUND: Trauma induces neutrophil migration toward injury sites, both initiating wound healing and protecting against local bacterial infection. We have previously shown that mitochondrial formyl peptides (mtFPs) released by injured tissues act as chemoattractants by ligating neutrophil (PMN) formyl peptide receptor 1 (FPR1). But this process can also internalize multiple neutrophil chemoattractant receptors and thus might limit neutrophil migration to the lung in response to bacteria. Our objective was to better understand susceptibility to pneumonia after injury and thus find ways to reverse it. METHODS AND RESULTS: We modeled the alveolar chemotactic environment in pulmonary infections by incubating Staphylococcus aureus or Escherichia coli with peripheral blood mononuclear cells. Survey of the chemotactic mediators in the resultant conditioned media (CM) showed multiple potent chemoattractants. Pretreating PMN with mtFPs to mimic injury potently reduced net migration toward CM and this net effect was mostly reversed by an FPR1 antagonist. Using an established mouse model of injury-dependent lung infection, we then showed simple instillation of exogenous unstimulated human neutrophils into the airway resulted in bacterial clearance from the lung. CONCLUSION: Injury-derived mtFPs suppress global PMN localization into complex chemotactic environments like infected alveoli. Transplantation of naive exogenous human neutrophils into the airway circumvents that pathologic process and prevents development of post-traumatic pneumonia without injury noted to the recipients.

Multiplexed Plasma Immune Mediator Signatures Can Differentiate Sepsis From NonInfective SIRS: American Surgical Association 2020 Annual Meeting Paper.

OBJECTIVES: Sepsis and sterile both release "danger signals' that induce the systemic inflammatory response syndrome (SIRS). So differentiating infection from SIRS can be challenging. Precision diagnostic assays could limit unnecessary antibiotic use, improving outcomes. METHODS: After surveying human leukocyte cytokine production responses to sterile damage-associated molecular patterns (DAMPs), bacterial pathogen-associated molecular patterns, and bacteria we created a multiplex assay for 31 cytokines. We then studied plasma from patients with bacteremia, septic shock, "severe sepsis," or trauma (ISS ≥15 with circulating DAMPs) as well as controls. Infections were adjudicated based on post-hospitalization review. Plasma was studied in infection and injury using univariate and multivariate means to determine how such multiplex assays could best distinguish infective from noninfective SIRS. RESULTS: Infected patients had high plasma interleukin (IL)-6, IL-1α, and triggering receptor expressed on myeloid cells-1 (TREM-1) compared to controls [false discovery rates (FDR) <0.01, <0.01, <0.0001]. Conversely, injury suppressed many mediators including MDC (FDR <0.0001), TREM-1 (FDR <0.001), IP-10 (FDR <0.01), MCP-3 (FDR <0.05), FLT3L (FDR <0.05), Tweak, (FDR <0.05), GRO-α (FDR <0.05), and ENA-78 (FDR <0.05). In univariate studies, analyte overlap between clinical groups prevented clinical relevance. Multivariate models discriminated injury and infection much better, with the 2-group random-forest model classifying 11/11 injury and 28/29 infection patients correctly in out-of-bag validation. CONCLUSIONS: Circulating cytokines in traumatic SIRS differ markedly from those in health or sepsis. Variability limits the accuracy of single-mediator assays but machine learning based on multiplexed plasma assays revealed distinct patterns in sepsis- and injury-related SIRS. Defining biomarker release patterns that distinguish specific SIRS populations might allow decreased antibiotic use in those clinical situations. Large prospective studies are needed to validate and operationalize this approach.

Formyl Peptide Receptor-1 Blockade Prevents Receptor Regulation by Mitochondrial Danger-Associated Molecular Patterns and Preserves Neutrophil Function After Trauma.

OBJECTIVES: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. DESIGN: Prospective study of human and murine neutrophils and clinical cohort analysis. SETTING: University research laboratory and level 1 trauma center. PATIENTS: Trauma patients, volunteer controls. ANIMAL SUBJECTS: C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. INTERVENTIONS: Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. MEASUREMENTS AND MAIN RESULTS: Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. CONCLUSIONS: Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.

Mitochondrial DNA in the tumour microenvironment activates neutrophils and is associated with worse outcomes in patients with advanced epithelial ovarian cancer.

BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.

Scavenging Circulating Mitochondrial DNA as a Potential Therapeutic Option for Multiple Organ Dysfunction in Trauma Hemorrhage.

Trauma is a leading cause of death worldwide with 5.8 million deaths occurring yearly. Almost 40% of trauma deaths are due to bleeding and occur in the first few hours after injury. Of the remaining severely injured patients up to 25% develop a dysregulated immune response leading to multiple organ dysfunction syndrome (MODS). Despite improvements in trauma care, the morbidity and mortality of this condition remains very high. Massive traumatic injury can overwhelm endogenous homeostatic mechanisms even with prompt treatment. The underlying mechanisms driving MODS are also not fully elucidated. As a result, successful therapies for trauma-related MODS are lacking. Trauma causes tissue damage that releases a large number of endogenous damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs released in trauma, such as mitochondrial DNA (mtDNA), could help to explain part of the immune response in trauma given the structural similarities between mitochondria and bacteria. MtDNA, like bacterial DNA, contains an abundance of highly stimulatory unmethylated CpG DNA motifs that signal through toll-like receptor-9 to produce inflammation. MtDNA has been shown to be highly damaging when injected into healthy animals causing acute organ injury to develop. Elevated circulating levels of mtDNA have been reported in trauma patients but an association with clinically meaningful outcomes has not been established in a large cohort. We aimed to determine whether mtDNA released after clinical trauma hemorrhage is sufficient for the development of MODS. Secondly, we aimed to determine the extent of mtDNA release with varying degrees of tissue injury and hemorrhagic shock in a clinically relevant rodent model. Our final aim was to determine whether neutralizing mtDNA with the nucleic acid scavenging polymer, hexadimethrine bromide (HDMBr), at a clinically relevant time point in vivo would reduce the severity of organ injury in this model. CONCLUSIONS: We have shown that the release of mtDNA is sufficient for the development of multiple organ injury. MtDNA concentrations likely peak at different points in the early postinjury phase dependent on the degree of isolated trauma vs combined trauma and hemorrhagic shock. HDMBr scavenging of circulating mtDNA (and nuclear DNA, nDNA) is associated with rescue from severe multiple organ injury in the animal model. This suggests that HDMBr could have utility in rescue from human trauma-induced MODS.

A subset of five human mitochondrial formyl peptides mimics bacterial peptides and functionally deactivates human neutrophils.

BACKGROUND: Trauma causes inflammation by releasing mitochondria that act as Danger-Associated Molecular Patterns (DAMPs). Trauma also increases susceptibility to infection. Human mitochondria contain 13 N-formyl peptides (mtFPs). We studied whether mtFPs released into plasma by clinical injury induce neutrophil (PMN) inflammatory responses, whether their potency reflects their similarity to bacterial FPs and how their presence at clinically relevant concentration affects PMN function. METHODS: N-terminal sequences of the 13 mtFPs were synthesized. Changes in human PMN cytosolic Ca concentration ([Ca]i) and chemotactic responses to mtFPs were studied. Sequence similarity of mtFPs to the canonical bacterial peptide f-Met-Leu-Phe (fMLF/fMLP) was studied using the BLOcks SUbstitution Matrix 62 (BLOSUM 62) system. The presence of mtFPs in plasma of trauma patients was assayed by Enzyme-linked immunosorbent assay (ELISA). The effects of the most potent mtFP (ND6) on PMN signaling and function were then studied at ambient clinical concentrations by serial exposure of native PMN to ND6, chemokines and leukotrienes. RESULTS: Five mtFPs (ND6, ND3, ND4, ND5, and Cox 1) induced [Ca]i flux and chemotaxis in descending order of potency. Evolutionary similarity to fMLF predicted [Ca]i flux and chemotactic potency linearly (R = 0.97, R = 0.95). Chemoattractant potency was also linearly related to [Ca]i flux induction (R = 0.92). Active mtFPs appear to circulate in significant amounts immediately after trauma and persist through the first week. The most active mtFP, ND6, suppresses responses to physiologic alveolar chemoattractants (CXCL-1, leukotriene B4) as well as to fMLF where CXCL-1 and leukotriene B4 do not suppress N-formyl peptide receptor (FPR)-1 responses to mtFPs. Prior FPR-1 inhibition rescues PMN from heterologous suppression of CXCR-1 and BLT-1 by mtFPs. CONCLUSION: The data suggest mtFPs released by injured tissue may attract PMN to trauma sites while suppressing PMN responses to other chemoattractants. Inhibition of mtFP-FPR1 interactions might increase PMN recruitment to lung bacterial inoculation after trauma. These findings suggest new paradigms for preventing infections after trauma. LEVEL OF EVIDENCE: Therapeutic, Level IV.

Mitochondrial DAMPs Are Released During Cardiopulmonary Bypass Surgery and Are Associated With Postoperative Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most frequent complication of surgery performed on cardiopulmonary bypass (CPB) and recent work associates CPB with postoperative inflammation. We have shown that all tissue injury releases mitochondrial damage associated molecular patterns (mtDAMPs) including mitochondrial DNA (mtDNA). This can act as a direct, early activator of neutrophils (PMN), eliciting a systemic inflammatory response syndrome (SIRS) while suppressing PMN function. Neutrophil Extracellular Traps (NETs) are crucial to host defence. They carry out NETosis wherein webs of granule proteins and chromatin trap and kill bacteria. We hypothesised that surgery performed on CPB releases mtDAMPs into the circulation. Molecular patterns thus mobilised during CPB might then participate in the pathogenesis of SIRS and predict postoperative complications like AF [1]. METHODS: We prospectively studied 16 patients undergoing elective operations on CPB. Blood was sampled preoperatively, at the end of CPB and on days 1-2 postoperatively. Plasma samples were analysed for mtDNA. Neutrophil IL-6 gene expression was studied to assess induction of SIRS. Neutrophils were also assayed for the presence of neutrophil extracellular traps (NETs/NETosis). These biologic findings were then correlated to clinical data and compared in patients with and without postoperative AF (POAF). RESULTS: Mitochondrial DNA was significantly elevated following CPB (six-fold increase post-CPB, p=0.008 and five-fold increase days 1-2, p=0.02). Patients with POAF showed greater increases in mtDNA post-CPB than those without. Postoperative AF was seen in all patients with a ≥2-fold increase of mtDNA (p=0.037 vs. <2-fold). Neutrophil IL-6 gene transcription increased postoperatively demonstrating SIRS that was greatest days 1-2 (p=0.039). Neutrophil extracellular trap (NET) formation was markedly suppressed in the post-CPB state. CONCLUSION: Mitochondrial DNA is released by CPB surgery and is associated with POAF. IL-6 gene expression increases after CPB, demonstrating the evolution of postoperative SIRS. Lastly, cardiac surgery on CPB also suppressed PMN NETosis. Taken together, our data suggest that mtDNA released during surgery on CPB, may be involved in the pathogenesis of SIRS and related postoperative inflammatory events like POAF and infections. Mitochondrial DNA may therefore prove to be an early biomarker for postoperative complications with the degree of association to be determined in appropriately sized studies. If mtDNA is directly involved in cardiac inflammation, mtDNA-induced toll-like receptor-9 (TLR9) signalling could also be targeted therapeutically.

Intratracheal instillation of neutrophils rescues bacterial overgrowth initiated by trauma damage-associated molecular patterns.

BACKGROUND: Nosocomial pneumonias are common in trauma patients and so interventions to prevent and treat nosocomial pneumonia may improve outcomes. Our prior work strongly suggests that tissue injury predisposes to infections like nosocomial pneumonia because mitochondrial debris originating from injured cells contains damage-associated molecular patterns that can reduce neutrophil (PMN) migration into the airway and diminish PMN function in response to bacterial inoculation of the airway. This suggested that putting exogenous "normal" PMN into the airway might be beneficial. METHODS: Postinjury pneumonia (PNA) commonly arises in two groups, early, community-acquired PNA (CAP) and later hospital-acquired PNA (HAP). Posttraumatic early-onset CAP and late-onset HAP were modeled in CD-1 mice using Staphylococcus aureus or Pseudomonas aeruginosa instilled intratracheal (i.t.) at clinically relevant times with or without extrapulmonary injuries mimicked by an intraperitoneal application of mitochondrial damage-associated molecular patterns. We applied bone marrow-derived PMN (BM-PMN) intratracheally to assess their effect on bacterial clearance in the lung. RESULTS: BM-PMN instillation i.t. had no untoward clinical effects on recipient animals. In both the early/CAP and late/HAP models, clearance of the bacterial inoculum from the lung was suppressed by mitochondrial debris and restored to uninjured levels by i.t. instillation of exogenous BM-PMN. Furthermore, PMN instillation cleared the inoculum of P. aeruginosa that could not be cleared by uninjured mice. Instillation of PMN into the lung, even across strains (CD-1 vs. C57BL/6) had no injurious effect. CONCLUSION: These initial studies suggest PMN instillation (i.t.) is worthy of further study as a potential adjunctive therapy aimed at decreasing the morbidity of lung infections in trauma patients. Moreover, PMN instillation (i.t.) may represent a unique means of preventing or treating pneumonia after serious injury that is completely independent of the need for antibiotic use.

A "CLEAN CASE" OF SYSTEMIC INJURY: MESENTERIC LYMPH AFTER HEMORRHAGIC SHOCK ELICITS A STERILE INFLAMMATORY RESPONSE.

Postinjury multiple organ failure results from an inappropriate overwhelming immune response to injury. During trauma and hemorrhagic shock (T/HS), mesenteric ischemia causes gut mucosal breakdown with disruption of the intestinal barrier. It has been proposed that this releases the gut microbiota systemically via postshock mesenteric lymph (PSML), engendering infectious complications. Despite extensive investigation, no clear evidence has been presented for gut bacterial translocation after resuscitation from T/HS. However, such previous studies were limited by available technologies. More sensitive methods, such as quantitative polymerase chain reaction, have since emerged for detection of bacterial presence and danger-associated molecular patterns (DAMPs). Quantitative polymerase chain reaction was applied to PSML derived from a rat model of T/HS. No bacterial presence was detected in a series of 12 samples, whereas multiple lymph samples showed the presence of DAMPs after T/HS. Thus, we confirmed that bacterial translocation does not exist in PSML after resuscitation from T/HS-associated mesenteric ischemia. However, T/HS does increase the presence of mitochondrial DAMPs in PSML. These results support our current position that PSML elaborates remote organ injury by multiple inflammatory mechanisms, including lipid-mediated proinflammatory stimuli, and by contribution from gut-derived DAMPs.

Pro-Coagulant Endothelial Dysfunction Results from EHEC Shiga Toxins and Host Damage-Associated Molecular Patterns.

Hemolytic uremic syndrome (HUS) from enterohemorrhagic Escherichia coli infection is a leading cause of kidney failure in otherwise healthy U.S. children. The bacterial Shiga toxins (Stx) induce the characteristic coagulopathy of HUS, but the damage to toxin-receptor expressing cells and organ injury due to ischemia likely also releases inflammatory damage-associated molecular patterns (DAMPs), which may exacerbate injury along with the toxins. To examine this, human aortic and renal glomerular cell anti-coagulant and barrier functions were studied after in vitro challenge with Stx1, Stx2, and DAMPs. There was significant loss of surface anti-coagulant protein C pathway molecules, increased expression of pro-thrombotic PAR1 and reduced protein C activation capability by 15-27%. Histones nearly completely prevented the activated protein C protection of endothelial cells from thrombin-induced permeability. In mice, lethal Stx2 challenge elevated plasma HMGB1 (day 2, 321 ± 118%; p < 0.01) and extracellular histones (day 3, 158 ± 62%; p < 0.01). Mice colonized with Stx2-expressing Citrobacter rodentium developed increased HMGB1 (day 5, 155 ± 55%; p < 0.01) and histones (day 3, 378 ± 188%; p < 0.01). Anti-histone antibody reduced both DAMPs to baseline, but was not sufficient to improve survival outcome or kidney function. Together, these data suggest a potential role Stx to produce DAMPs, and DAMPs to produce endothelial injury and a pro-thrombotic environment.

Mitochondrial DNA released by trauma induces neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are critical for anti-bacterial activity of the innate immune system. We have previously shown that mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA (mtDNA), are released into the circulation after injury. We therefore questioned whether mtDNA is involved in trauma-induced NET formation. Treatment of human polymorphoneutrophils (PMN) with mtDNA induced robust NET formation, though in contrast to phorbol myristate acetate (PMA) stimulation, no NADPH-oxidase involvement was required. Moreover, formation of mtDNA-induced NETs was completely blocked by TLR9 antagonist, ODN-TTAGGG. Knowing that infective outcomes of trauma in elderly people are more severe than in young people, we measured plasma mtDNA and NET formation in elderly and young trauma patients and control subjects. MtDNA levels were significantly higher in the plasma of elderly trauma patients than young patients, despite lower injury severity scores in the elderly group. NETs were not visible in circulating PMN isolated from either young or old control subjects. NETs were however, detected in PMN isolated from young trauma patients and to a lesser extent from elderly patients. Stimulation by PMA induced widespread NET formation in PMN from both young volunteers and young trauma patients. NET response to PMA was much less pronounced in both elderly volunteers' PMN and in trauma patients' PMN. We conclude that mtDNA is a potent inducer of NETs that activates PMN via TLR9 without NADPH-oxidase involvement. We suggest that decreased NET formation in the elderly regardless of higher mtDNA levels in their plasma may result from decreased levels of TLR9 and/or other molecules, such as neutrophil elastase and myeloperoxidase that are involved in NET generation. Further study of the links between circulating mtDNA and NET formation may elucidate the mechanisms of trauma-related organ failure as well as the greater susceptibility to secondary infection in elderly trauma patients.