An explorative study on deep profiling of peripheral leukocytes to identify predictors for responsiveness to anti-tumour necrosis factor alpha therapies in ankylosing spondylitis: natural killer cells in focus.

BACKGROUND: Therapeutic targeting of tumour necrosis factor (TNF)-α is highly effective in ankylosing spondylitis (AS) patients. However, since one-third of anti-TNF-treated AS patients do not show an adequate clinical response there is an urgent need for new biomarkers that would aid clinicians in their decision-making to select appropriate therapeutic options. Thus, the aim of this explorative study was to identify cell-based biomarkers in peripheral blood that could be used for a pre-treatment stratification of AS patients. METHODS: A high-dimensional, multi-parametric flow cytometric approach was applied to identify baseline predictors in 31 AS patients before treatment with the TNF blockers adalimumab (TNF-neutralisation) and etanercept (soluble TNF receptor). RESULTS: As the major result, the frequencies of natural killer (NK) cells, and in particular CD8-positive (CD8+) NK cell subsets, were most predictive for therapeutic outcome in AS patients. While an inverse correlation between classical CD56+/CD16+ NK cells and reduction of disease activity was observed, the CD8+ NK cell subset behaved in the opposite direction. At baseline, responders showed significantly increased frequencies of CD8+ NK cells compared with non-responders. CONCLUSIONS: This is the first study demonstrating that the composition of the NK cell compartment has predictive power for prediction of therapeutic outcome for anti-TNF-α blockers, and we identified CD8+ NK cells as a potential new player in the TNF-α-driven chronic inflammatory immune response of AS.

Defining TNF-α- and LPS-induced gene signatures in monocytes to unravel the complexity of peripheral blood transcriptomes in health and disease.

Several genome-wide transcriptome studies have shown that chronic inflammatory responses generally taking place in the inflamed tissue are also reflected at the level of peripheral blood leukocytes. Blood monocytes are highly sensitized cell type continuously activated under inflammatory conditions. For a better understanding of the transcriptional imprinting influenced by a multitude of pro- and anti-inflammatory mediators, we established a whole blood in vitro system to explore cell- and stimulus-specific gene expression signatures in peripheral monocytes. In an explorative study, whole blood from healthy donors was stimulated with tumour necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) for 1.5 h. Subsequently, monocytes were isolated with a purity of >99% by high-speed fluorescence activated cell sorting. Transcriptional changes were explored by whole genome Affymetrix arrays using highly validated filtering algorithm to identify differentially expressed genes. In vitro stimulation of whole blood samples with TNF-α and LPS resulted in 4,529 and 5,036 differentially expressed genes, respectively. Although both stimuli induced similar inflammatory profiles in monocytes, TNF-α- or LPS-specific gene signatures were characterized. Functional classification identified significant numbers of differentially expressed cytokines, cytokine receptors and apoptosis-associated genes. To our knowledge, this is the first study presenting cell- and stimulus-specific gene expression signatures that can be used to decipher complex disease specific profiles of acute and chronic inflammation. Once a library of signatures from the most important inflammatory mediators is defined, it can be helpful to identify those signatures, which are predominantly driving the disease pathogenesis and which are of potential interest for a therapeutical intervention.

Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus.

OBJECTIVE: Type I interferon (IFN) plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE) and is therefore considered a potential therapeutic target. This study was undertaken to establish a feasible biomarker for IFN effects with respect to disease activity and effectiveness of IFN-suppressive therapy in SLE patients. METHODS: Transcriptomes of purified monocytes from 9 SLE patients and 7 healthy controls were analyzed by Affymetrix GeneChip technology. Levels of sialic acid-binding Ig-like lectin 1 (Siglec-1) (sialoadhesin, CD169) in inflammatory and resident monocytes were determined at the protein level in 38 healthy controls and 52 SLE patients, using multicolor flow cytometry. RESULTS: Transcriptomes of peripheral monocytes from SLE patients revealed a dominant type I IFN signature. Siglec-1 was identified as one of the most prominent type I IFN-regulated candidate genes. At the protein level, the frequency of Siglec-1-expressing monocyte subsets was correlated with disease activity (as measured by the SLE Disease Activity Index) and was inversely correlated with levels of complement factors. Most interestingly, levels of anti-double-stranded DNA (anti-dsDNA) antibodies were highly correlated with the percentage of resident monocytes, but not inflammatory monocytes, expressing Siglec-1. High-dose glucocorticoid treatment resulted in a dramatic reduction of Siglec-1 expression in cells from patients with active SLE. CONCLUSION: Our findings indicate that Siglec-1 expression in resident blood monocytes is a potential biomarker for monitoring disease activity, displaying type I IFN responses, and estimating levels of anti-dsDNA antibodies. Moreover, our results suggest that resident and inflammatory monocytes contribute differently to the process of autoantibody formation in SLE.

From transcriptome to cytome: integrating cytometric profiling, multivariate cluster, and prediction analyses for a phenotypical classification of inflammatory diseases.

Gene expression studies of peripheral blood cells in inflammatory diseases revealed a large array of new antigens as potential biomarkers useful for diagnosis, prognosis, and therapy stratification. Generally, their validation on the protein level remains mainly restricted to a more hypothesis-driven manner. State-of-the-art multicolor flow cytometry make it attractive to validate candidate genes at the protein and single cell level combined with a detailed immunophenotyping of blood cell subsets. We developed multicolor staining panels including up to 50 different monoclonal antibodies that allowed the assessment of several hundreds of phenotypical parameters in a few milliliters of peripheral blood. Up to 10 different surface antigens were measured simultaneously by the combination of seven different fluorescence colors. In a pilot study blood samples of ankylosing spondylitis (AS) patients were compared with normal donors (ND). A special focus was set on the establishment of suitable bioinformatic strategy for storing and analyzing hundreds of phenotypical parameters obtained from a single blood sample. We could establish a set of multicolor stainings that allowed monitoring of all major leukocyte populations and their corresponding subtypes in peripheral blood. In addition, antigens involved in complement and antibody binding, cell migration, and activation were acquired. The feasibility of our cytometric profiling approach was demonstrated by a successful classification of AS samples with a reduced subset of 80 statistically significant parameters, which are partially involved in antigen presentation and cell migration. Furthermore, these parameters allowed an error-free prediction of independent AS and ND samples originally not included for parameter selection. This study demonstrates a new level of multiparametric analysis in the post-transcriptomic era. The integration of an appropriate bioinformatic solution as presented here by the combination of a custom-made Access database along with cluster- and prediction-analysis tools predestine our approach to promote the human cytome project.