Kim Timmermans

The anti-CD20 antibody rituximab reduces the Th17 cell response

OBJECTIVE Rituximab has been shown to be successful in the treatment of rheumatoid arthritis (RA), and this unexpected finding indicates that B cells have an important role in this disease. The present study was undertaken to investigate the mechanism of action of rituximab in RA. METHODS Twelve patients with active RA were treated with rituximab. Disease activity was evaluated using the 28-joint Disease Activity Score. Synovial biopsy samples obtained at baseline and 12 weeks after treatment initiation were analyzed by microarray, quantitative polymerase chain reaction, and immunohistochemistry. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and from 4 patients with X-linked agammaglobulinemia were stimulated with the Th17-inducing stimulus Candida albicans, and the response in the presence and absence of rituximab was examined. RESULTS In RA patients, rituximab reduced expression of retinoic acid-related orphan receptor γt and interleukin-22 (IL-22) and numbers of Th17-positive cells in synovial tissue, and this correlated with better clinical outcome. Rituximab did not affect tumor necrosis factor α (TNFα), Th1 cell, or Treg cell responses. Rituximab strongly reduced in vitro IL-17 and IL-22 production induced by C albicans. This effect was not observed in PBMCs from patients with X-linked agammaglobulinemia. CONCLUSION Rituximab reduced the local Th17 response in RA patients, whereas it did not influence Th1 cell, Treg cell, or TNFα responses. The decreased Th17 response was associated with reduced inflammation and better clinical outcome. Moreover, inhibition of the Th17 response by rituximab was lost in the absence of B cells, providing evidence that the effects of rituximab are due to B cell depletion. These data demonstrate an unexpected role of B cells in the development of Th17 responses, which could possibly lead to B cell-based strategies for the treatment of Th17-related autoimmune diseases.

DANGER IN THE INTENSIVE CARE UNIT: DAMPS IN CRITICALLY ILL PATIENTS

ABSTRACT Danger-associated molecular patterns (DAMPs) that are released by injured, threatened, or dead cells, or that originate from the extracellular matrix, influence the immune system. This is of great relevance in critically ill patients, in whom trauma or surgery-related cell damage, hypoxia, ischemia, and infections can result in extensive release of DAMPs. As many patients at the intensive care unit suffer from immune system-related complications, DAMPs could serve as markers for the prognosis of these patients and represent possible therapeutic targets. In the present review, we provide an overview of several well known DAMPs (high-mobility group box 1, heat-shock proteins, s100 proteins, nucleic acids, and hyaluronan) and their effects on the immune system. Furthermore, we discuss the role of DAMPs as markers or therapeutic targets in several conditions frequently encountered in critically ill patients, such as sepsis, trauma, ventilator-induced lung injury, and cardiac arrest.

Blueprints of signaling interactions between pattern recognition receptors: implications for the design of vaccine adjuvants.

ABSTRACT Innate immunity activation largely depends on recognition of microorganism structures by Pattern Recognition Receptors (PRRs). PRR downstream signaling results in production of pro- and anti-inflammatory cytokines and other mediators. Moreover, PRR engagement in antigen-presenting cells initiates the activation of adaptive immunity. Recent reports suggest that for the activation of innate immune responses and initiation of adaptive immunity, synergistic effects between two or more PRRs are necessary. No systematic analysis of the interaction between the major PRR pathways were performed to date. In this study, a systematical analysis of the interactions between PRR signaling pathways was performed. PBMCs derived from 10 healthy volunteers were stimulated with either a single PRR ligand or a combination of two PRR ligands. Known ligands for the major PRR families were used: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RigI-helicases. After 24 h of incubation, production of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and IL-10 was measured in supernatants by enzyme-linked immunosorbent assay (ELISA). The consistency of the PRR interactions (both inhibitory and synergistic) between the various individuals was assessed. A number of PRR-dependent signaling interactions were found to be consistent, both between individuals and with regard to multiple cytokines. The combinations of TLR2 and NOD2, TLR5 and NOD2, TLR5 and TLR3, and TLR5 and TLR9 acted as synergistic combinations. Surprisingly, inhibitory interactions between TLR4 and TLR2, TLR4 and Dectin-1, and TLR2 and TLR9 as well as TLR3 and TLR2 were observed. These consistent signaling interactions between PRR combinations may represent promising targets for immunomodulation and vaccine adjuvant development.

Plasma Nuclear and Mitochondrial DNA Levels, and Markers of Inflammation, Shock, and Organ Damage in Patients with Septic Shock

Background: Plasma levels of the danger-associated molecular patterns (DAMPs) nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have been shown to be related to sepsis mortality. However, the intermediate factors and/or mechanisms contributing to this relation are largely unknown. Our aim was to determine whether plasma levels of nDNA and mtDNA are related to the markers of inflammation, severity of shock, and organ damage in septic shock patients. Moreover, we investigated the relationship between plasma levels of nDNA/mtDNA and inflammatory cytokines during experimental human endotoxemia, a model of systemic inflammation in humans in vivo mimicking some of the hallmarks of early sepsis. Methods: Blood was sampled from the onset of septic shock until day 28 in 121 septic shock patients and from 1 h before endotoxin administration until 8 h afterward in 12 healthy volunteers. Plasma concentrations of five cytokines and circulating levels of nDNA and mtDNA were measured, and correlations with shock-related parameters and markers of organ damage were investigated. Results: In septic shock patients plasma cytokine concentrations, as well as nDNA and mtDNA levels, were increased at the onset of septic shock and remained elevated. During the first 5 days of septic shock, nDNA levels consistently correlated with plasma cytokine concentrations as well as with the shock-related parameter norepinephrine infusion rate and markers of organ damage (total bilirubin and creatinine). Experimental human endotoxemia also resulted in increased levels of plasma nDNA and mtDNA, but to a lesser extent than in septic shock patients. Furthermore, nDNA levels correlated with pro-inflammatory cytokines during endotoxemia. Conclusions: Our findings indicate a relationship between plasma nDNA levels and the inflammatory response. Furthermore, nDNA levels are associated with markers of shock and organ damage in septic shock patients. Nevertheless, the correlations found are relatively weak and it remains to be determined whether nDNA is merely a marker or directly involved in the pathophysiology of septic shock.

The Involvement of Danger-associated Molecular Patterns in the Development of Immunoparalysis in Cardiac Arrest Patients

Objectives: After cardiac arrest, patients are highly vulnerable toward infections, possibly due to a suppressed state of the immune system called “immunoparalysis.” We investigated if immunoparalysis develops following cardiac arrest and whether the release of danger-associated molecular patterns could be involved. Design: Observational study. Setting: ICU of a university medical center. Patients: Fourteen post–cardiac arrest patients treated with mild therapeutic hypothermia for 24 hours and 11 control subjects. Measurements and Main Results: Plasma cytokines showed highest levels within 24 hours after cardiac arrest and decreased during the next 2 days. By contrast, ex vivo production of cytokines interleukin-6, tumor necrosis factor-&agr;, and interleukin-10 by lipopolysaccharide-stimulated leukocytes was severely impaired compared with control subjects, with most profound effects observed at day 0, and only partially recovering afterward. Compared with incubation at 37°C, incubation at 32°C resulted in higher interleukin-6 and lower interleukin-10 production by lipopolysaccharide-stimulated leukocytes of control subjects, but not of patients. Plasma nuclear DNA, used as a marker for general danger-associated molecular pattern release, and the specific danger-associated molecular patterns (EN-RAGE and heat shock protein 70) were substantially higher in patients at days 0 and 1 compared with control subjects. Furthermore, plasma heat shock protein 70 levels were negatively correlated with ex vivo production of inflammatory mediators interleukin-6, tumor necrosis factor-&agr;, and interleukin-10. Extracellular newly identified receptor for advanced glycation end products-binding protein levels only showed a significant negative correlation with ex vivo production of interleukin-6 and tumor necrosis factor-&agr; and a borderline significant inverse correlation with interleukin-10. No significant correlations were observed between plasma nuclear DNA levels and ex vivo cytokine production. Interventions: None. Conclusions: Release of danger-associated molecular patterns during the first days after cardiac arrest is associated with the development of immunoparalysis. This could explain the increased susceptibility toward infections in cardiac arrest patients.

Circulating iFABP Levels as a marker of intestinal damage in trauma patients

ABSTRACT Both the initial trauma and the subsequent hemodynamic instability may contribute to intestinal damage, which is of great importance in (immunological) posttrauma complications. This study assesses intestinal damage using the biomarker intestinal Fatty Acid Binding Protein (iFABP) in trauma patients during the first days of their hospital admission and the risk factors involved. Plasma iFABP levels were measured in blood samples obtained from adult multiple trauma patients (n = 93) at the trauma scene by the Helicopter Emergency Medical Services, at arrival at the emergency department (ED), and at days 1, 3, 5, 7, 10, and 14 after trauma and related to injury severity and hemodynamic parameters. Plasma iFABP concentrations showed highest levels immediately after trauma at time points Helicopter Emergency Medical Services and ED. Nonsurvivors demonstrated higher iFABP levels at the ED compared with survivors. Furthermore, iFABP values at the ED correlated with Injury Severity Scores, and patients suffering from abdominal trauma demonstrated significantly higher iFABP concentrations in comparison with patients with other types of trauma or healthy controls. Also, patients presenting with a mean arterial pressure (MAP) less than 70 mmHg at the ED demonstrated significantly higher plasma iFABP concentrations in comparison with patients with a normal (70–99 mmHg) or high (>100 mmHg) MAP or healthy controls. Finally, patients with a low hemoglobin (Hb) (<80% of reference value) displayed significantly higher iFABP concentrations in comparison with patients with a normal Hb or healthy controls. Plasma iFABP levels, indicative of intestinal injury, are increased immediately after trauma in patients with abdominal trauma, low MAP, or low Hb and are related to the severity of the trauma. As intestinal injury is suggested to be related to late complications, such as multiorgan dysfunction syndrome or sepsis in trauma patients, strategies to prevent intestinal damage after trauma could be of benefit to these patients.

Mitochondrial DNA and TLR9 Signaling Is Not Involved in Mechanical Ventilation-Induced Inflammation.

Exogenous administration of mitochondrial DNA (mtDNA) causes inflammatory lung injury in a toll-like receptor (TLR) 9-dependent manner. We investigated whether mechanical ventilation results in endogenous release of mtDNA and whether TLR9 plays a role in the pulmonary inflammatory response induced by mechanical ventilation.Wild-type and TLR9−/− C57bl/6 mice were ventilated with low (8 mL/kg) and high (32 mL/kg) tidal volumes for 4 hours. Levels of nuclear DNA and mtDNA in bronchoalveolar lavage fluid, as well as pulmonary concentrations of keratinocyte-derived chemokine, interleukin-1&bgr;, and interleukin-6, were determined.Cytokine and nuclear DNA, but not mtDNA, levels were increased after mechanical ventilation with both tidal volumes. Cytokine concentrations were similar between wild-type and TLR9−/− mice. Mechanical ventilation does not result in the release of mtDNA, and TLR9 is not involved in mechanical ventilation-induced inflammation.

Release of Danger-Associated Molecular Patterns following Chemotherapy Does Not Induce Immunoparalysis in Leukemia Patients

Chemotherapy may result in the release of danger-associated molecular patterns (DAMPs), which can cause immunoparalysis (deactivation of the immune system). We investigated DAMPs following chemotherapy and their relationship with markers of immunoparalysis in leukemia patients. In 6 patients with acute myeloid leukemia or myelodysplastic syndrome and 12 healthy subjects, DAMPs, cytokines, and markers of immunoparalysis were determined before and during the first week after chemotherapy initiation. In the patients, plasma levels of nuclear DNA (a marker of general DAMP release) were profoundly increased before chemotherapy and further increased 4-6 h afterwards, while the specific DAMP mitochondrial DNA showed only a trend towards increase. Circulating cytokine levels did not change following chemotherapy. Leukocyte cytokine production capacity and HLA-DR expression were similar in patients and healthy controls until day 4 when leukocytes were found to be virtually absent. In conclusion, in the early phase following chemotherapy in leukemia patients, increased DAMP release does not induce immunoparalysis.

Soluble urokinase-type plasminogen activator levels are related to plasma cytokine levels but have low predictive value for mortality in trauma patients

INTRODUCTION Soluble urokinase-type plasminogen activator (suPAR) represents a marker for immune activation and has predictive value in critically ill patients. The kinetics of suPAR and its correlation with the immune response and outcome in trauma patients are unknown. METHODS Plasma concentrations of inflammatory cytokines and suPAR were determined in adult trauma patient (n = 69) samples obtained by the Helicopter Emergency Medical Services at arrival at the emergency department (ED) and at days 1, 3, 5, 7, 10, and 14. RESULTS Initial suPAR levels were unrelated to injury severity score and higher in nonsurvivors compared with survivors, although no difference was observed between early and late mortality. The area under the receiver operating characteristic curve to predict mortality was 0.6 (95% confidence interval, 0.48-0.72). Soluble urokinase-type plasminogen activator levels increased over time in 94% of patients, although suPAR increase did not precede death. Tumor necrosis factor α at the ED correlated with suPAR at that time point, whereas concentrations of other proinflammatory cytokines at the ED correlated with suPAR levels at days 1 and 5. CONCLUSIONS After trauma, initial suPAR plasma concentrations are higher in nonsurvivors compared with survivors, but its predictive value is low. Soluble urokinase-type plasminogen activator levels increase over time after trauma, and concentrations at later time points are related to cytokine levels at the ED.

IL-1β processing in mechanical ventilation-induced inflammation is dependent on neutrophil factors rather than caspase-1

PurposeMechanical ventilation can cause ventilator-induced lung injury, characterized by a sterile inflammatory response in the lungs resulting in tissue damage and respiratory failure. The cytokine interleukin-1β (IL-1β) is thought to play an important role in the pathogenesis of ventilator-induced lung injury. Cleavage of the inactive precursor pro-IL-1β to form bioactive IL-1β is mediated by several types of proteases, of which caspase-1, activated within the inflammasome, is the most important. Herein, we studied the roles of IL-1β, caspase-1 and neutrophil factors in the mechanical ventilation-induced inflammatory response in mice.MethodsUntreated wild-type mice, IL-1αβ knockout and caspase-1 knockout mice, pralnacasan (a selective caspase-1 inhibitor)-treated mice, anti-keratinocyte-derived chemokine (KC)-treated mice and cyclophosphamide-treated neutrophil-depleted wild-type mice were ventilated using clinically relevant ventilator settings (tidal volume 8 ml/kg). The lungs and plasma were collected to determine blood gas values, cytokine profiles and neutrophil influx.ResultsMechanical ventilation resulted in increased pulmonary concentrations of IL-1β and KC and increased pulmonary neutrophil influx compared with non-ventilated mice. Ventilated IL-1αβ knockout mice did not demonstrate this increase in cytokines. No significant differences were observed between wild-type and caspase-1-deficient or pralnacasan-treated mice. In contrast, in anti-KC antibody-treated mice and neutropenic mice, inflammatory parameters decreased in comparison with ventilated non-treated mice.ConclusionsOur results illustrate that IL-1 is indeed an important cytokine in the inflammatory cascade induced by mechanical ventilation. However, the inflammasome/caspase-1 appears not to be involved in IL-1β processing in this type of inflammatory response. The attenuated inflammatory response observed in ventilated anti-KC-treated and neutropenic mice suggests that IL-1β processing in mechanical ventilation-induced inflammation is mainly mediated by neutrophil factors.