Azithromycin inhibits IL-1 secretion and non-canonical inflammasome activation

Deregulation of inflammasome activation was recently identified to be involved in the pathogenesis of various inflammatory diseases. Although macrolide antibiotics display well described immunomodulatory properties, presumably involved in their clinical effects, their impact on inflammasome activation has not been investigated. We compared the influence of macrolides on cytokine induction in human monocytes. The role of intracellular azithromycin- accumulation was examined by interference with Ca++-dependent uptake. We have also analysed the signalling cascades involved in inflammasome activation, and substantiated the findings in a murine sepsis model. Azithromycin, but not clarithromycin or roxithromycin, specifically inhibited IL-1α and IL-1β secretion upon LPS stimulation. Interference with Ca++-dependent uptake abolished the cytokine-modulatory effect, suggesting a role of intracellular azithromycin accumulation in the modulatory role of this macrolide. Azithromycin’s inhibiting effects were observed upon LPS, but not upon flagellin, stimulation. Consistent with this observation, we found impaired induction of the LPS-sensing caspase-4 whereas NF-κB signalling was unaffected. Furthermore, azithromycin specifically affected IL-1β levels in a murine endotoxin sepsis model. We provide the first evidence of a differential impact of macrolides on the inflammasome/IL-1β axis, which may be of relevance in inflammasome-driven diseases such as chronic obstructive pulmonary disease or asthma

Testing lupus anticoagulants in a real-life scenario - a retrospective cohort study

Introduction: Lupus anticoagulant (LAC) testing is challenging. Most data are derived from a well-controlled study environment with potential alterations to daily routines. The aim of this retrospective cohort study was to assess the capacity of various LAC screening tests and derived mixing tests to predict a positive result in subsequent confirmation tests in a large cohort of patients. Materials and methods: In 5832 individuals, we retrospectively evaluated the accuracy of the aPTT-A, aPTT-LAscreen, aPTT-FS and dRVVTscreen and of their derived mixing tests in detecting a positive confirmation test result within the same blood specimen. The group differences, degree of correlation and the predictive accuracy of LAC coagulation tests were analysed using the Mann-Whitney U test, the Spearman-rank-correlation and by area under the receiver operating characteristic curve (ROC-AUC) analysis. ROC-AUCs were compared with the Venkatraman´s permutation test. Results: The pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LAscreen showed the best diagnostic accuracy with a ROC-AUC of 0.84 (95% CI: 0.82 – 0.86). No clear advantage of the dRVVT-derived mixing test was detectable when compared to the dRVVTscreen (P = 0.829). Usage of the index of circulating anticoagulant (ICA) did not improve the diagnostic power of respective mixing tests. Conclusions: Among the parameters evaluated, aPTT-LAscreen and derived mixing test parameters were the most accurate tests. In our study cohort, neither other mixing test nor the ICA presented any further advantage in LAC diagnostics

Scientific Reports / Azithromycin inhibits IL-1 secretion and non-canonical inflammasome activation

Deregulation of inflammasome activation was recently identified to be involved in the pathogenesis of various inflammatory diseases. Although macrolide antibiotics display well described immunomodulatory properties, presumably involved in their clinical effects, their impact on inflammasome activation has not been investigated. We compared the influence of macrolides on cytokine induction in human monocytes. The role of intracellular azithromycin-accumulation was examined by interference with Ca++-dependent uptake. We have also analysed the signalling cascades involved in inflammasome activation and substantiated the findings in a murine sepsis model. Azithromycin, but not clarithromycin or roxithromycin, specifically inhibited IL-1 and IL-1 secretion upon LPS stimulation. Interference with Ca++-dependent uptake abolished the cytokine-modulatory effect, suggesting a role of intracellular azithromycin accumulation in the modulatory role of this macrolide. Azithromycins inhibiting effects were observed upon LPS, but not upon flagellin, stimulation. Consistent with this observation, we found impaired induction of the LPS-sensing caspase-4 whereas NF-B signalling was unaffected. Furthermore, azithromycin specifically affected IL-1 levels in a murine endotoxin sepsis model. We provide the first evidence of a differential impact of macrolides on the inflammasome/IL-1 axis, which may be of relevance in inflammasome-driven diseases such as chronic obstructive pulmonary disease or asthma.(VLID)491092

Evaluation of the Septifast MGrade Test on Standard Care Wards--A Cohort Study.

BACKGROUND:The immediate need for appropriate antimicrobial therapy in septic patients requires the detection of the causative pathogen in a timely and reliable manner. In this study, the real-time PCR Septifast MGrade test was evaluated in adult patients meeting the systemic inflammatory response syndrome (SIRS) criteria that were treated at standard care wards. METHODS:Patients with clinical suspected infection, drawn blood cultures (BC), the Septifast M(Grade) test (SF) and sepsis biomarkers were prospectively screened for fulfillment of SIRS criteria and evaluated using the criteria of the European Centre of Disease Control (ECDC) for infection point prevalence studies. RESULTS:In total, 220 patients with SIRS were prospectively enrolled, including 56 patients with detection of bacteria in the blood (incidence: 25.5%). BC analysis resulted in 75.0% sensitivity (95% confidence interval, CI: 61.6%- 85.6%) with 97.6% specificity (CI: 93.9%- 99.3%) for detecting bacteria in the blood. In comparison to BC, SF presented with 80.4% sensitivity (CI: 67.6%- 89.8%) and with 97.6% specificity (CI: 93.9%- 99.3%). BC and SF analysis yielded comparable ROC-AUCs (0.86, 0.89), which did not differ significantly (p = 0.558). A trend of a shorter time-to-positivity of BC analysis was not seen in bacteremic patients with a positive SF test than those with a negative test result. Sepsis biomarkers, including PCT, IL-6 or CRP, did not help to explain discordant test results for BC and SF. CONCLUSION:Since negative results do not exclude bacteremia, the Septifast M(Grade) test is not suited to replacing BC, but it is a valuable tool with which to complement BC for faster detection of pathogens

Strong CD8⁺ T cell effector functions induced by IL-2::2Ig(F)GPI asVNP.

<p>(<b>A</b>) Flow cytometry analysis showing proliferation and intracellular IFN-γ-expression of CFSE-labeled CD8^<b>+</b> TCR Vα2^<b>+</b> T-cells. Markers set according to negative stainings and no-stimulation control. Numbers indicate percentage of cells in respective quadrants. (<b>B</b>) Flow cytometry analysis, (<b>C</b>) percentage and (<b>D</b>) fold-induction of IFN-γ producing CD8^<b>+</b> T cells in the absence or presence of indicated stimuli. (<b>E</b>) Amounts of IFN-γ (pg/ml) secreted upon co-culture of CD8^<b>+</b> T cells with indicated IL-2v asVNP or αCD3/αCD28 coated microbeads plus IL-2 (10 or 100 U/ml). (<b>F-H</b>) IL-2::2Ig(F)GPI asVNP increase the cytotoxic potential of antigen-specific T cells. P14 splenocytes were stimulated with IL-2v asVNP for 48 hours or left untreated and were subsequently co-cultured with 1x10^<b>4</b> wt (CPD^{<b>bright</b>}) or LCMV-GP expressing (CPD^<b>dim</b>) EL-4 target cells. Specific target cell lysis was analyzed after 20 hours by flow cytometry. (<b>F</b>) Flow cytometry analysis showing specific lysis of LCMV-GP^<b>+</b> but not wt target cells at an effector/target ratio of 10. (<b>G</b>) Quantification by flow cytometric analysis of specific target cell lysis relative to maximum lysis (LCMV-GP_33-41 stimulated effector cells, E:T ratio 20:1). (<b>H</b>) Determination of cytotoxic potential in ^<b>51</b>Cr-release assays of P14 splenocytes, which were stimulated with IL-2v asVNP or left untreated for 48 hours. Effector cells were co-cultured with ^<b>51</b>Cr-labelled wildtype or LCMV-GP-expressing EL-4 target cells (1x10^<b>4</b>) and specific lysis was analyzed. Data are representative (A, B, F, H) or show the summary (C, D, E, G) of three (A, B, F, G), five (except three for αCD3/αCD28 microbeads) (C, D), four (except two for 48 and 72 hrs) (E) and triplicates of one (H) independent experiments. * p < 0.05, **, p < 0.01, ***, p < 0.001; ANOVA and Tukey’s multiple comparison test (C, D), Kruskal-Wallis test and Mann-Whitney U-test and Bonferroni correction (E), t-test comparing IL-2::GPI with IL-2::2Ig(F)GPI asVNP (G, H).</p

Differential activation of CD8⁺ T cells by IL-2v decorated VNP.

<p>(<b>A</b>) Flow cytometry analysis of CD69 and TCR expression on P14 TCR transgenic splenocytes. Cells were either left untreated (grey shaded histograms), or stimulated (black line) with IL-2v asVNP or alternatively with αCD3/αCD28 coated microbeads supplemented with 10 or 100 U/ml IL-2 for 18 hours. Percentages of CD69^<b>high</b> and TCR^<b>low</b> cells are shown. (<b>B</b> and <b>C</b>) Flow cytometry analysis of CD25 expression on CD8^<b>+</b> T cells. Purified P14 CD8^<b>+</b> T cells were stimulated for the indicated number of days with IL-2v asVNP, or αCD3/αCD28 coated microbeads supplemented with 10 or 100 U/ml IL-2, as indicated or were left untreated. (<b>B</b>) CD25 expression on CD8^<b>+</b> T cells co-cultured with IL-2v asVNP for three days. Numbers indicate MFI of CD25^<b>+</b> cells. (<b>C</b>) Kinetics of CD25 expression on CD8^<b>+</b> T cells stimulated with indicated asVNP, ansVNP, or αCD3/αCD28 coated microbeads supplemented with 10 or 100 U/ml IL-2. (<b>D</b>) Proliferation of CD8^<b>+</b> T cells upon incubation with IL-2v asVNP, ansVNP, medium or PMA/ionomycin. (<b>E</b>) Absolute numbers of viable P14 CD8^<b>+</b> T cells after 5 days of co-culture with IL-2v asVNP or αCD3/αCD28 coated microbeads supplemented with IL-2. Data are representative (A, B) or show the summary (C-E) of four (except two for αCD3/αCD28) (A), five (except three for ansVNP) (B), at least three (C), nine (except five for ansVNP and two αCD3/αCD28 microbeads) (D) and seven (except four for αCD3/αCD28 microbeads) (E) independent experiments. * p < 0.05, **, p < 0.01, ***, p < 0.001; t-test comparing IL-2::GPI with IL-2::2Ig(F)GPI asVNP (C), Kruskal-Wallis test and Mann-Whitney U-test followed by post hoc Bonferroni correction (D) ANOVA and Tukey’s multiple comparison test (E).</p

CD8⁺ T Cell Fate and Function Influenced by Antigen-Specific Virus-Like Nanoparticles Co-Expressing Membrane Tethered IL-2

<div><p>A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here determined whether systemically toxic cytokines such as IL-2 can be restricted to the site of antigen presentation and used as ‘natural adjuvants’. Therefore, we devised antigen-presenting virus-like nanoparticles (VNP) co-expressing IL-2 attached to different membrane-anchors and assessed their potency to modulate CD8⁺ T cell responses <i>in vitro</i> and <i>in vivo</i>. Efficient targeting of IL-2 to lipid rafts and ultimately VNP was achieved by fusing IL-2 at its C-terminus to a minimal glycosylphosphatidylinositol (GPI)-anchor acceptor sequence. To identify optimal membrane-anchor dimensions we inserted one (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b between IL-2 and the minimal GPI-anchor acceptor sequence of CD16b (GPI). We found that the 2IgGPI version was superior to all other evaluated IL-2 variants (IL-2v) in terms of its i) degree of targeting to lipid rafts and to the VNP surface, ii) biological activity, iii) co-stimulation of cognate T cells in the absence of bystander activation and iv) potency to induce differentiation and acquisition of CD8⁺ T cell effector functions <i>in vitro</i> and <i>in vivo</i>. In contrast, the GPI version rather favored memory precursor cell formation. These results exemplify novel beneficial features of membrane-bound IL-2, which in addition to its mere T cell stimulatory capacity include the induction of differential effector and memory functions in CD8⁺ T lymphocytes.</p></div