Sepsis-induced long-term immune paralysis – results of a descriptive, explorative study

Background: Long-lasting impairment of the immune system is believed to be the underlying reason for delayed deaths after surviving sepsis. We tested the hypothesis of persisting changes to the immune system in survivors of sepsis for the first time. Methods: In our prospective, cross-sectional pilot study, eight former patients who survived catecholamine-dependent sepsis and eight control individuals matched for age, sex, diabetes and renal insufficiency were enrolled. Each participant completed a questionnaire concerning morbidities, medications and infection history. Peripheral blood was collected for determination of i) immune cell subsets (CD4+, CD8+ T cells; CD25+ CD127- regulatory T cells; CD14+ monocytes), ii) cell surface receptor expression (PD-1, BTLA, TLR2, TLR4, TLR5, Dectin-1, PD-1 L), iii) HLA-DR expression, and iv) cytokine secretion (IL-6, IL10, TNF-α, IFN-γ) of whole blood stimulated with either α-CD3/28, LPS or zymosan. Results: After surviving sepsis, former patients presented with increased numbers of clinical apparent infections, including those typically associated with an impaired immune system. Standard inflammatory markers indicated a low-level inflammatory situation in former sepsis patients. CD8+ cell surface receptor as well as monocytic HLA-DR density measurements showed no major differences between the groups, while CD4+ T cells tended towards two opposed mechanisms of negative immune cell regulation via PD-1 and BTLA. Moreover, the post-sepsis group showed alterations in monocyte surface expression of distinct pattern recognition receptors; most pronouncedly seen in a decrease of TLR5 expression. Cytokine secretion in response to important activators of both the innate (LPS, zymosan) and the adaptive immune system (α-CD3/28) seemed to be weakened in former septic patients. Conclusions: Cytokine secretion as a reaction to different activators of the immune system seemed to be comprehensively impaired in survivors of sepsis. Among others, this could be based on trends in the downregulation of distinct cell surface receptors. Based on our results, the conduct of larger validation studies seems feasible, aiming to characterize alterations and to find potential therapeutic targets to engage

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner.The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA

Impact of human sepsis on CCCTC-binding factor associated monocyte transcriptional response of Major Histocompatibility Complex II components.

BACKGROUND:Antigen presentation on monocyte surface to T-cells by Major Histocompatibility Complex, Class II (MHC-II) molecules is fundamental for pathogen recognition and efficient host response. Accordingly, loss of Major Histocompatibility Complex, Class II, DR (HLA-DR) surface expression indicates impaired monocyte functionality in patients suffering from sepsis-induced immunosuppression. Besides the impact of Class II Major Histocompatibility Complex Transactivator (CIITA) on MHC-II gene expression, X box-like (XL) sequences have been proposed as further regulatory elements. These elements are bound by the DNA-binding protein CCCTC-Binding Factor (CTCF), a superordinate modulator of gene transcription. Here, we hypothesized a differential interaction of CTCF with the MHC-II locus contributing to an altered monocyte response in immunocompromised septic patients. METHODS:We collected blood from six patients diagnosed with sepsis and six healthy controls. Flow cytometric analysis was used to identify sepsis-induced immune suppression, while inflammatory cytokine levels in blood were determined via ELISA. Isolation of CD14++ CD16-monocytes was followed by (i) RNA extraction for gene expression analysis and (ii) chromatin immunoprecipitation to assess the distribution of CTCF and chromatin modifications in selected MHC-II regions. RESULTS:Compared to healthy controls, CD14++ CD16-monocytes from septic patients with immune suppression displayed an increased binding of CTCF within the MHC-II locus combined with decreased transcription of CIITA gene. In detail, enhanced CTCF enrichment was detected on the intergenic sequence XL9 separating two subregions coding for MHC-II genes. Depending on the relative localisation to XL9, gene expression of both regions was differentially affected in patients with sepsis. CONCLUSION:Our experiments demonstrate for the first time that differential CTCF binding at XL9 is accompanied by uncoupled MHC-II expression as well as transcriptional and epigenetic alterations of the MHC-II regulator CIITA in septic patients. Overall, our findings indicate a sepsis-induced enhancer blockade mediated by variation of CTCF at the intergenic sequence XL9 in altered monocytes during immunosuppression

Sepsis induces specific changes in histone modification patterns in human monocytes.

Sepsis is a global burden and the primary cause of death in intensive care units worldwide. The pathophysiological changes induced by the host's systemic inflammatory response to infection are not yet fully understood. During sepsis, the immune system is confronted with a variety of factors, which are integrated within the individual cells and result in changes of their basal state of responsiveness. Epigenetic mechanisms like histone modifications are known to participate in the control of immune reactions, but so far the situation during sepsis is unknown.In a pilot approach, we performed combined chromatin immunoprecipitation followed by high-throughput sequencing to assess the genome-wide distribution of the chromatin modifications histone 3 lysine 4 and 27 trimethylation and lysine 9 acetylation in monocytes isolated from healthy donors (n = 4) and patients with sepsis (n = 2). Despite different underlying causes for sepsis, a comparison over promoter regions shows a high correlation between the patients for all chromatin marks. These findings hold true also when comparing patients to healthy controls. Despite the global similarity, differential analysis reveals a set of distinct promoters with significant enrichment or depletion of histone marks. Further analysis of overrepresented GO terms show an enrichment of genes involved in immune function. To the most prominent ones belong different members of the HLA family located within the MHC cluster together with the gene coding for the major regulator of this locus-CIITA.We are able to show for the first time that sepsis in humans induces selective and precise changes of chromatin modifications in distinct promoter regions of immunologically relevant genes, shedding light on basal regulatory mechanisms that might be contributing to the functional changes occurring in monocytes

Experimental design/workflow.

<p>Diagram shows the experimental design for a complete workflow to analyse genome wide histone modifications of CD 14++ CD 16- monocytes using 30 ml of blood.</p

Histone modifications at promoters of CD 14++ CD 16- monocytes.

<p>A) H3K4me3 and H3K27me3 are both enriched at promoters. Significantly bound regions were compared with the distribution of genomic features (TSS, TSS upstream, TES, exon, intron, intergenic) based on RefSeq gene annotations. In comparison to the genomic background (genome) both H3K27me3, but especially H3K4me3 are strongly enriched for TSSs. B) K-means clustering reveals 3 major chromatin states at 44109 RefSeq transcriptional start sites. The heat map shows binding (in red) centered across a 10 kb interval spanning all RefSeq TSSs for H3K27me3 and H3K4me3 in 4 blood donors. The data was clustered by k-means which separates the data into 3 major promoter classes: no histone modification (dark blue), high H3K4me3/no H3K27me3 (blue) and low but detectable H3K4me3 and high H3K4me3 (light blue). C) Promoter classes are associated with specific transcriptional output. Gene expression levels in CD14+ monocytes were calculated for all RefSeq genes (based on ENCODE RNA-seq data, shown as reads per 1 kb of transcript length). Gene expression values of genes falling into above mentioned categories are plotted as boxplots.</p

Correlation analysis shows high reproducibility between independent biological samples.

<p>A) H3K4me3 binding data from 4 donors was compared against each other with respect to binding to promoters. Quantile-normalized read counts per promoter interval (+/−1 kb around the transcriptional start sites) were plotted for all pair-wise combinations as smoothed scatterplots (color-coding of density is shown in color key). Furthermore Pearson’s R was calculated for each combination. B) Pair wise correlation was done for H3K4me3, H3K27me3 as well as H3K9ac data from 4 donors. The hierarchical cluster analysis of the resulting correlation matrix identifies two major groups: H3K9ac and H3K4me3 are strongly correlated whereas both are negatively correlated with H3K27me3 (color-coding of correlation coefficients is shown in color key).</p

Figure 1

<p><b>A, B:</b> Microscopy for PTV measurements. Section of microscopic top view on the tracheal epithelium between 2 cartilages for PTV measurement (20x lens). <b>A:</b> Arrows indicate the applied particles (Dynabeads) <b>B:</b> Recorded particle tracks for PTV calculation. <b>C:</b> Representative experimental setup: The stimulation phase started 60 minutes after organ explantation. Measurements were performed over a period of 60 minutes (stimulation period). Vitality of the ciliary cells was tested by ATP application (10⁻⁵ M) over 10 minutes (vitality test) (Control group, n = 5, Mean ± S.E.M). <b>D:</b> Microscopy for CBF measurements<b>.</b> Section of microscopic top view between 2 cartilages for CBF measurement (40x lens)<b>.</b> Encircled area shows cilia bearing epithelial cell.</p