Cytokines are peptide hormones playing a major role in regulating both acute pathogen-induced inflammation and chronic inflammation. In general, amount and balance of pro- and anti-inflammatory cytokines controls severity and duration of acute and chronic inflammation. Furthermore, it is assumed that a stable inbalance of pro-inflammatory and anti-inflammatory cytokines could contribute critically to the transition from acute to chronic inflammation. The importance of cytokines in acute inflammation has been demonstrated by the lethal effects of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-#) in experimental septic shock. Furthermore, targeted knockout of cytokine genes in mice has demonstrated that the normally benign response to commensal gut microflora turns into a lethal inflammation, in the absence of the cytokines interleukin 2 (IL-2)or interleukin 10 (IL-10). IL-2 or IL-2 receptor deficient mice revealed the non-redundant role of IL-2 in the control of peripheral T cell tolerance driven by regulatory T cells.

Here, we are performing global transcriptome analysis of ex vivo isolated and in vitro treated cells to better understand molecular mechanisms of acute and chronic inflammation, indicate pathways of immunopathology and potential drug targets, identify surrogate markers for cytometric and serological profiling, and discover markers for prognosis of therapeutic success.

The particular goals of this project are:
(i) to identify cell type-specific cytokine and anti-cytokine (therapeutics) gene expression signatures,induced by inflammation-related cytokines in monocytes and T helper cells. Combined induction of cytokine-signaling and cell specific activation will provide a more comprehensive view of the interactive complex signaling networks.
(ii) to examine gene expression signatures in induced in ex vivo isolated cells in close collaboration with other groups of NGFNII and with pathogens of interest for the consortium e.g. Yersiniae, Filariea, H. pylori and M. tuberculosis. Comparison of cytokine and pathogen induced signatures will help to elucidate pathogenic pathways critical for development of inflammation.
(iii) to analyse the function of several genes identified already in the frame of NGFN I, as genes expressed specifically in cytokine-polarized, chronically activated murine T helper lym-phocytes, and in acute activated human T helper lymphocytes and monocytes. These are potential drug targets or/and markers for cytometric profiling.
(iv) to deliver reference signatures of cytokine, pathogen, and therapeutic treatment and compare these signatures with in vivo induced cell-signatures in mice and men in acute and chronic inflammation examined in different projects within the network. These will indicate pathways of immunopathology and potential drug targets, identify surrogate markers for cytometric and serological profiling, and for prognosis of therapeutic success.

Project Status

Cytokine gene expression signatures
Cytokines act particularly via modulating the activation and differentiation of T helper cells and monocytes. To detect direct action of cytokines on gene expression in these cells, we performed global gene expression analysis of ex vivo isolated T helper cells and monocytes of healthy donors triggered in vitro through the cytokine receptors. To better understand physiological and pathological mechanisms of cytokine action we also combined induction of cytokine-signaling and cell specific activation.
To date, we studied the effects of the cytokines IL-2 and TGF-ß in T-cells and TNF-# in monocytes. To prepare the global transcriptom analysis we first estimated the kinetics of
• TCR stimulation and IL-2 expression in CD4+ T cells,
• TGF-ß treatment of CD4+ T cells with and without TCR stimulation, and
• TNF-# treatment of monocytes.
Second, we established the IL-2 secretion procedure to obtain pure populations of T cells with the property either to produce IL-2 or not.

Initial analysis of the gene expression data from IL-2 producing and IL-2 non-producing CD4+ T cells showed that 1120 genes are differentially expressed 2h after TCR-stimulation (filter conditions were: signal height > 100 and fold change > 1.6). Further experiments will emerge if some of these genes/proteins are causal responsible for the ability of T cells to produce IL-2 or not.

Using RT-PCR (light cycler) we evaluated the gene expression of 4 TGF-ß induced genes in murine T cells. The isolated pure CD4+ T cells were treated with 0.1 – 2 ng/ml TGF-ß concentrations with or without TCR stimulation for 1.5 – 16 h. We evaluated the following time points as most relevant for global transcriptom analysis: 1.5, 4, 8, and 16 h. The best concentrations to reflect low and high TGF-ß level were 0.1 and 2 ng/ml TGF-ß, respectively.

In collaboration with Andreas Grützkau and Thomas Häupl (other projects within the NGFN II) and Joachim Gruen (Oligene GmbH) we compared the in vitro TNF-# induced gene expression signatures with rheumatic disease specific in vivo signatures. We identified several common genes. Further validation and functional studies will show if few of these are target genes for disease treatment and/or of relevance for diagnosis and/or prognosis of therapeutic success.

Functional analysis of selected genes/proteins
To better understand molecular mechanisms of cytokine function in inflammation we continued our work of NGFN I and other projects. In order to understand the molecular basis of induction of and memory for expression of cytokine genes in T lymphocytes controling chronic immune reactions, we determined global gene-expression profiles of in vitro cytokine-polarized, chronically activated and acutely activated murine and human T helper lymphocytes. We have already detected several specifically expressed genes, which we are analysing functionally. So far, there are 5 genes of outstanding importance.
In particular, we studied the function of one gene expressed specifically in acute activated human T helper lymphocytes. Furthermore this candidate is an immediate early gene. We found out that this gene plays a key role in the induction of regulatory T cells and therefore in tolerance induction.

Outlook
Cytokines are controling chronic inflammation, and, in concert with pathogen-derived signals, also acute inflammation. Here, cytokine- and pathogen-induced gene expression profiles will be obtained from normal human and murine monocytes and T helper lymphocytes, isolated ex vivo according to SOP developed in NGFN I, and confronted in vitro with the respective inducing signal. Beside IL-2, TGF ß, and TNF-# also type I and II IFN, IL-10, IL-1, IL-6, and IL 4, the pathogens of interest for the consortium, e.g. Yersiniae, Filariea, H. pylori, M. tuberculosis, and the drugs used clinically to treat chronic inflammation will be compared. The cell-type-specific, ex vivo gene expression signatures will reveal the direct effects of the cytokines, pathogens, and biologicals used to treat chronic rheumatic inflammation. These signatures will indicate pathways of immuno-pathology, identify surrogate markers for cytometric and serological profiling, and for prognosis of therapeutic success. Finally, they will be key to understand the monocyte and T helper cell-signatures induced in vivo in mice and men, in acute and chronic inflammation, which have been determined so far in NGFN I and are expected in NGFN II. We will continue to analyse several genes functionally, which we have identified already in the frame of NGFN I, as genes expressed specifically in cytokine-polarized, chronically activated murine T helper lymphocytes and immediate early genes of acute activated human T helper lymphocytes and monocytes.