S100A8/A9 mRNA induction in an ex vivo model of endotoxin tolerance: roles of IL-10 and IFNγ.

OBJECTIVES: Septic syndromes are the leading cause of death in intensive care units. They are characterized by the development of immune dysfunctions such as endotoxin tolerance (ET), whose intensity and duration are associated with increased risk of nosocomial infections and mortality. Alarmins S100A8 and S100A9 have been shown to be increased after septic shock. Importantly, a delayed S100A9 mRNA increase predicts hospital-acquired infection in patients. The aim of this study was to investigate the regulation of S100A8 and S100A9 mRNA expression in an ex vivo model of ET. SUBJECTS AND MEASUREMENTS: ET was reproduced ex vivo by priming healthy peripheral blood mononuclear cells (number of donors  = 9 to 10) with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. MAIN RESULTS: ET was established by observing decreased TNFα and increased IL-10 transcriptomic responses to two subsequent endotoxin challenges. Interestingly, ET was associated with increased S100A8 and S100A9 mRNA expression ex vivo. We showed that IL-10 played a role in this process, since S100A8 and S100A9 mRNA increases were significantly abrogated by IL-10 blockade in the model. Conversely, treatment with rIFN-γ, a pro-inflammatory and immunostimulating molecule known to block ET induction, was able to restore normal S100A8 and S100A9 mRNA in this model. CONCLUSIONS: In this ex vivo model, we observed that S100A8 and S100A9 mRNA expression was significantly increased during ET. This reproduced ex vivo the observations we had previously made in septic shock patients. Interestingly, IL-10 blockade and rIFN-γ treatment partially abrogated S100A8/A9 mRNA increases in this model. Pending confirmation in larger, independent clinical studies, these preliminary results suggest that S100A8 and S100A9 mRNA levels might be used as surrogate markers of ET and as stratification tools for personalized immunotherapy in septic shock patients.

Identification of biomarkers of response to IFNg during endotoxin tolerance: application to septic shock.

The rapid development in septic patients of features of marked immunosuppression associated with increased risk of nosocomial infections and mortality represents the rational for the initiation of immune targeted treatments in sepsis. However, as there is no clinical sign of immune dysfunctions, the current challenge is to develop biomarkers that will help clinicians identify the patients that would benefit from immunotherapy and monitor its efficacy. Using an in vitro model of endotoxin tolerance (ET), a pivotal feature of sepsis-induced immunosuppression in monocytes, we identified using gene expression profiling by microarray a panel of transcripts associated with the development of ET which expression was restored after immunostimulation with interferon-gamma (IFN-γ). These results were confirmed by qRT-PCR. Importantly, this short-list of markers was further evaluated in patients. Of these transcripts, six (TNFAIP6, FCN1, CXCL10, GBP1, CXCL5 and PID1) were differentially expressed in septic patients' blood compared to healthy blood upon ex vivo LPS stimulation and were restored by IFN-γ. In this study, by combining a microarray approach in an in vitro model and a validation in clinical samples, we identified a panel of six new transcripts that could be used for the identification of septic patients eligible for IFNg therapy. Along with the previously identified markers TNFa, IL10 and HLA-DRA, the potential value of these markers should now be evaluated in a larger cohort of patients. Upon favorable results, they could serve as stratification tools prior to immunostimulatory treatment and to monitor drug efficacy.

Importance of correlation between gene expression levels: application to the type I interferon signature in rheumatoid arthritis.

BACKGROUND: The analysis of gene expression data shows that many genes display similarity in their expression profiles suggesting some co-regulation. Here, we investigated the co-expression patterns in gene expression data and proposed a correlation-based research method to stratify individuals. METHODOLOGY/PRINCIPAL FINDINGS: Using blood from rheumatoid arthritis (RA) patients, we investigated the gene expression profiles from whole blood using Affymetrix microarray technology. Co-expressed genes were analyzed by a biclustering method, followed by gene ontology analysis of the relevant biclusters. Taking the type I interferon (IFN) pathway as an example, a classification algorithm was developed from the 102 RA patients and extended to 10 systemic lupus erythematosus (SLE) patients and 100 healthy volunteers to further characterize individuals. We developed a correlation-based algorithm referred to as Classification Algorithm Based on a Biological Signature (CABS), an alternative to other approaches focused specifically on the expression levels. This algorithm applied to the expression of 35 IFN-related genes showed that the IFN signature presented a heterogeneous expression between RA, SLE and healthy controls which could reflect the level of global IFN signature activation. Moreover, the monitoring of the IFN-related genes during the anti-TNF treatment identified changes in type I IFN gene activity induced in RA patients. CONCLUSIONS: In conclusion, we have proposed an original method to analyze genes sharing an expression pattern and a biological function showing that the activation levels of a biological signature could be characterized by its overall state of correlation.

mRNA-based approach to monitor recombinant gamma-interferon restoration of LPS-induced endotoxin tolerance.

INTRODUCTION: It is now well accepted that sepsis is associated with the development of a pronounced immunosuppressive state, characterized by severe immune alterations (e.g. reduced proliferative capacity, endotoxin tolerance, apoptosis) participating in increased mortality and susceptibility to nosocomial infections. Efforts are currently aimed at restoring a functional immune response in septic patients. Successful therapy depends on the identification of appropriate immunostimulatory drugs and on the development of suitable biomarkers that could be used to stratify patients and to follow response to treatment. METHODS: In this study, we evaluated the ex vivo effect of recombinant interferon gamma (rIFN-γ) in restoring monocyte functionality (endotoxin-induced Tumor Necrosis Factor-α production) in a two-hit model of endotoxin tolerance (ET) with peripheral blood mononuclear cells from healthy volunteers and in whole blood of septic shock patients. Importantly, we used quantitative-reverse transcription polymerase-chain reaction to monitor the effect of rIFN-γ on the expression of seven genes known to participate in ET (TNF-α, IL-10, HLA-DRA, CIITA, IRAK-M, ABIN-3 and LY64). RESULTS: Expression analysis of those genes confirmed the presence of an immunosuppression state and the ex vivo restoration of immune functions by rIFN-γ. We show for the first time that rIFN-γ is able to bypass, at the mRNA level, the effect of negative regulators of the LPS signalling pathway such as IRAK-M, ABIN-3 and LY64. CONCLUSIONS: Overall, mRNA expressions of a panel of genes could represent promising candidates for the ex vivo evaluation of rIFN-γ effect on monocyte functionality. This ex vivo translational research study demonstrates the potential of a mRNA-based approach to successfully monitor drug efficacy.

Upregulation of the pro-apoptotic genes BID and FAS in septic shock patients.

INTRODUCTION: Lymphocyte apoptosis has been suggested to play a central role in sepsis pathophysiology, and studies in animal models demonstrated that blocking this pathway improves outcome. However, no routine biomarkers of apoptosis are so far available in patients. Thus, the aim of our study was to assess the different biomarkers of apoptosis putatively usable on a routine basis in septic shock. METHODS: Thirteen septic shock patients (sampled twice between days 1 to 2 and days 3 to 5 after diagnosis of shock) and 15 sex-matched and age-matched healthy controls were prospectively enrolled. Apoptosis was measured in lymphocyte subpopulations using flow cytometry (Annexin-V binding, activated caspase-3 and Bcl-2 expressions). Representative pro-apoptotic and anti-apoptotic gene expressions were assessed by quantitative reverse-transcription PCR. Monocyte HLA-DR expression and lymphocyte subpopulation cell counts were measured as markers of sepsis-induced immune dysfunctions. To test for statistical significance, the Mann-Whitney U test was used with correction by the number of tests performed. RESULTS: Flow cytometric measurements of apoptosis in septic shock patients showed an increased Annexin-V binding on CD4+ T cells and an increased active caspase-3 expression on B cells only at days 3 to 5 (sixfold change and twofold change, respectively). Gene expression analysis showed an increased BCL-XL mRNA and an upregulation of the pro-apoptotic genes BID and FAS in septic shock patients (10-fold change and fivefold change, respectively) compared with healthy controls. CONCLUSIONS: The present study highlights the difficulties encountered in monitoring apoptosis on a routine basis in septic patients, whereas in the same sampling conditions and on the same patients, HLA-DR expression and lymphocyte subpopulation cell counts showed characteristics described in the literature. However, pro-apoptotic genes BID and FAS appear to constitute promising apoptosis markers in our hands.

FoxO3a involved in neutrophil and T cell survival is overexpressed in rheumatoid blood and synovial tissue.

OBJECTIVE: FoxO3a is a transcriptional factor implicated in cell cycle regulation and apoptosis. Since rheumatoid arthritis (RA) is associated with apoptosis defects, the expression level, regulation and phosphorylation status of FoxO3a was investigated in blood and synovium from patients with RA. METHODS: In microarray experiments, an overexpression of FoxO3a mRNA was observed in blood from patients with RA compared with healthy controls. FoxO3a mRNA expression was quantified in polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells from patients with RA by qRT-PCR. Total FoxO3a and phosphorylated FoxO3a (pFoxO3a) protein expression was analysed in blood leucocytes from patients with RA versus controls and in synovium from patients with RA versus patients with osteoarthritis (OA) by immunostaining. RESULTS: FoxO3a mRNA and protein expression levels were increased in blood from patients with RA compared with controls. FoxO3a overexpression was primarily observed in PMNs. In synovium from patients with RA, both total and inactive phosphorylated FoxO3a proteins were detected. FoxO3a was detected primarily in the sublining T lymphocytes of synovium from patients with RA compared with the lining layer tissue from patients with RA and OA, underlying a role for FoxO3a proteins in inflammation in RA. CONCLUSION: The overexpression of FoxO3a in blood from patients with RA, particularly in PMNs, suggests a potential role for this gene in the pathogenesis of RA through increased survival of blood PMNs. In synovium from patients with RA, FoxO3a mainly detected in inflammatory aggregates may also regulate the chronic survival of T lymphocytes.