Der Einfluß des BMP-Antagonisten Noggin auf die in vitro-Chondrogenese mesenchymaler Stammzellen

Sportlich und alters- bzw. degenerativ bedingter Verschleiß der hyalinen Knorpelkappen der Gelenke gehört heutzutage zu den häufigsten Erkrankungen weltweit, die sich hieraus ergebenden volkswirtschaftlichen Schäden sind enorm. Aufgrund seiner anatomischen Beschaffenheit und einer hiermit einhergehenden, im Vergleich zu anderen Gewebearten äußerst schlechten Regenerationsfähigkeit, ist einmal beschädigter Gelenkknorpel kaum mehr in der Lage, sich selbständig zu regenerieren, körpereigene Reparaturprozesse führen in der Regel lediglich zu minderwertigem Faserknorpel, der den mechanischen Anforderungen in den Gelenken nicht genügt. Auch die momentan angewendeten chirurgischen Maßnahmen bieten derzeit noch nicht die Möglichkeit, dem natürlichen Gelenkknorpel vergleichbare, funktionell hochwertige Ersatzgewebe zu generieren und den Patienten eine dauerhafte Beschwerdebesserung zu gewährleisten. Im Bereich des Tissue Engeneering haben sich in den letzten Jahren humane mesenchymale Stammzellen aufgrund ihrer relativ einfachen Isolierungsmöglichkeit sowie ihrer Fähigkeit, unter anderem chondrogen zu differenzieren, als vielversprechende Zellquelle für eine eventuelle Entwicklung derartiger Ersatzgewebe herausgestellt. Diese chondrogene Differenzierung kann in vitro in unterschiedlichen Chondrogenesemodellen induziert und untersucht werden, jedoch hat sich hier gezeigt, dass chondrogen differenzierende MSCs neben charakteristischen chondrogenen Markern wie Kollagen Typ II auch hypertrophieassoziierte Marker wie Kollagen Typ X und ALP exprimieren. Dies lässt vermuten, dass die chondrogene Differenzierung der MSCs in vitro vergleichbaren regulatorischen Mechanismen und Entwicklungsphasen folgt, wie sie in der endochondralen Ossifikation während der embryonalen Skelettentwicklung gefunden wird, in deren Verlauf die Chondrozyten hypertrophieren, schlussendlich apoptotisch werden und das Gewebe ossifiziert. Für die Herstellung von Knorpelersatzprodukten zur Behandlung von Knorpeldefekten ist diese Entwicklung jedoch höchst bedenklich, da die Voraussetzung hierfür die Stabilisierung des chondrogenen Phänotyps ist und hypertrophierende Zellen für derartige Ersatzgewbe ungeeignet sind. Insbesondere den BMPs (bone morphogenetic proteins) kommt dabei im komplexen Zusammenspiel einer Vielzahl unterschiedlicher Regelkreise eine entscheidende Rolle bei der Induktion der Hypertrophie zu. Ziel dieser Studie war die Untersuchung des BMP-Antagonisten Noggin, bzw. der Einfluß von Noggin auf die chondrogen differenzierenden MSCs in vitro und insbesondere inwieweit die Hypertrophie in diesen Zellen in Anwesenheit von Noggin im Kulturmedium beeinflusst bzw. unterdrückt werden kann. Hierzu wurden die MSCs in einem Pelletkulturmodell in einem definierten Medium mit u.a. TGF-ß und Dexamethason 14 Tage lang chondrogen vordifferenziert, im Anschluß wurde in einem Teil der Zellen durch Entzug von TGF-ß und Dexamethason sowie Zugabe des Schilddrüsenhormones T3 zum Medium die Hypertrophie angebahnt. Weiterhin wurden sowohl die in chondrogenem Medium belassenen, als auch die in hypertrophes Medium überführten Aggregate mit unterschiedlich hohen Dosen Noggin behandelt. Nach weiteren 14 Tagen wurden die Zellen dann histologisch untersucht, weiterhin wurde die Genexpression bestimmter Marker mittels PCR ermittelt. Die gewonnenen Ergebnisse zeigen deutlich, daß Noggin in der Lage ist, die T3-induzierte Hypertrophie in MSCs in vitro dosisabhängig zu inhibieren. Zusammenfassend weist dies darauf hin, daß die Beeinflußung des BMP-Signalweges mittels extrazellulärer BMP-Antagonisten wie Noggin ein möglicher Weg zur Verhinderung der Hypertrophie in der in vitro-Chondrogenese von MSCs und damit die Basis für die Herstellung MSC-basierter Knorpelersatzprodukte sein kann

Aberrant plasma IL-7 and soluble IL-7 receptor levels indicate impaired T-cell response to IL-7 in human tuberculosis

<div><p>T-cell proliferation and generation of protective memory during chronic infections depend on Interleukin-7 (IL-7) availability and receptivity. Regulation of IL-7 receptor (IL-7R) expression and signalling are key for IL-7-modulated T-cell functions. Aberrant expression of soluble (s) and membrane-associated (m) IL-7R molecules is associated with development of autoimmunity and immune failure in acquired immune deficiency syndrome (AIDS) patients. Here we investigated the role of IL-7/IL-7R on T-cell immunity in human tuberculosis. We performed two independent case-control studies comparing tuberculosis patients and healthy contacts. This was combined with follow-up examinations for a subgroup of tuberculosis patients under therapy and recovery. Blood plasma and T cells were characterised for IL-7/sIL-7R and mIL-7R expression, respectively. IL-7-dependent T-cell functions were determined by analysing STAT5 phosphorylation, antigen-specific cytokine release and by analysing markers of T-cell exhaustion and inflammation. Tuberculosis patients had lower soluble IL-7R (p < 0.001) and higher IL-7 (p < 0.001) plasma concentrations as compared to healthy contacts. Both markers were largely independent and aberrant expression normalised during therapy and recovery. Furthermore, tuberculosis patients had lower levels of mIL-7R in T cells caused by post-transcriptional mechanisms. Functional <i>in vitro</i> tests indicated diminished IL-7-induced STAT5 phosphorylation and impaired IL-7-promoted cytokine release of <i>Mycobacterium tuberculosis</i>-specific CD4⁺ T cells from tuberculosis patients. Finally, we determined T-cell exhaustion markers PD-1 and SOCS3 and detected increased SOCS3 expression during therapy. Only moderate correlation of PD-1 and SOCS3 with IL-7 expression was observed. We conclude that diminished soluble IL-7R and increased IL-7 plasma concentrations, as well as decreased membrane-associated IL-7R expression in T cells, reflect impaired T-cell sensitivity to IL-7 in tuberculosis patients. These findings show similarities to pathognomonic features of impaired T-cell functions and immune failure described in AIDS patients.</p></div

Membrane-associated IL-7R expression on CD4⁺ and CD8⁺ T cells.

<p>Membrane-associated IL-7R expression of CD4⁺ (left graphs) and CD8⁺ (right graphs) T cells from tuberculosis patients (n = 17) and healthy contacts (n = 21) analysed by flow cytometry. <b>(a)</b> Mean fluorescence intensity (MFI) analyses of IL-7R. <b>(b)</b> Proportions of IL-7R_low-expressing CD4⁺ and CD8⁺ T cells from tuberculosis patients and healthy contacts. Median and interquartile range is depicted, and each symbol indicates mean values of duplicates from each individual donor. Exact Mann-Whitney U test used for comparison of groups. Nominal p-values are indicated as * p < 0.05, ** p < 0.01.</p

Plasma IL-7 concentration of tuberculosis patients and healthy contacts.

<p><b>(a)</b> Comparison of IL-7 plasma concentrations between tuberculosis patients (n = 52) and healthy contacts (n = 148). <b>(b)</b> Plasma IL-7 concentrations prior to (0 months, n = 52), during (2 months, n = 46), and after (6 months, n = 41) treatment of tuberculosis patients. Median IL-7 plasma level of healthy contacts is represented with a dotted line. <b>(c)</b> Spearman correlation between plasma IL-7 and sIL-7R for tuberculosis patients prior to treatment (squares), and healthy contacts (circles). <b>(d)</b> Receiver operating characteristic (ROC) curve indicates sensitivity and specificity of plasma sIL-7R (solid line) and plasma IL-7 (dashed line) to discriminate between patients with tuberculosis and healthy contacts. The line of no discrimination is indicated as a dotted line. Median and interquartile range is depicted, and each symbol indicates mean values of duplicates from each individual donor. Exact Mann-Whitney U test was used for comparison of groups, whereas paired data was evaluated by Wilcoxon Signed-Rank test. Nominal p-values are indicated as: * p < 0.05, *** p < 0.001.</p

PD-1 and SOCS3 mRNA expression of CD4+ T cells from tuberculosis patients and healthy contacts.

<p>The expression of PD-1 <b>(a)</b> and SOCS3 <b>(c)</b> was determined for mRNA isolated from CD4⁺ T cells, using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference. Cycle threshold differences (2^-ΔCt) are shown for healthy contacts [n = 117 (PD-1), n = 119 (SOCS3)], and for tuberculosis patients prior to (0 months, n = 40), during (2 months, n = 28), and after (6 months, n = 17) treatment. Median and interquartile range is depicted. Spearman correlation between plasma IL-7 and <b>(b)</b> PD-1 or <b>(d)</b> SOCS3 for healthy contacts (circles) or tuberculosis patients (squares) prior to treatment. Each symbol indicates mean values of duplicates from each individual donor. Due to a low overlap between tuberculosis patients, exact Mann-Whitney U test used for comparison of all groups. Nominal p-values are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Patient characteristics.

<p>Patient characteristics.</p

IL-7 response of CD4⁺ T cells from tuberculosis patients and healthy contacts.

<p><b>(a)</b> IL-7-induced (10 ng/ml) STAT5 phosphorylation of CD4⁺ T cells from tuberculosis patients (n = 22) and healthy contacts (n = 24) measured by flow cytometry. The STAT5 phosphorylation level of non-stimulated cells has been subtracted of all values. <b>(b)</b> IFNγ/CD40L-expressing CD4⁺ T cells after PPD re-stimulation in the presence or absence of IL-7 detected by flow cytometry. Non-stimulated values with or without IL-7 have been subtracted. <b>(c)</b> Induction of IFNγ/CD40L-expressing CD4⁺ T cells by IL-7 and PPD stimulation. Absolute differences as compared to PPD alone are shown. <b>(d)</b> Plasma IL-6 levels of tuberculosis patients (n = 20) and healthy contacts (n = 24). An arbitrary threshold indicated by a dotted line was set to define tuberculosis patients with high (IL-6_high) and low (IL-6_low) concentrations of plasma IL-6. <b>(e)</b> STAT5 phosphorylation, and IL-7-induced PPD response <b>(f)</b> for tuberculosis patients with high or low plasma IL-6 level, as defined in (d). Median and interquartile range is depicted, and exact Mann-Whitney U test was applied for comparison of groups, whereas paired data was evaluated by Wilcoxon Signed-Rank test. Nominal p-values are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Plasma sIL-7R level in healthy contacts and tuberculosis during chemotherapy.

<p><b>(a)</b> Concentrations of sIL-7R in plasma from tuberculosis patients (n = 52) and healthy contacts (n = 149) determined by cytometric bead array. <b>(b)</b> sIL-7R plasma concentration prior to (0 months, n = 52), during (2 months, n = 46) and after (6 months, n = 41) treatment of tuberculosis patients. Median sIL-7R plasma concentration of healthy contacts is indicated with a dotted line. <b>(c)</b> Plasma sIL-7R levels stratified for the <i>IL7RA</i> exon 6 single nucleotide polymorphism rs6897932C>T for healthy contacts (n = 142) and tuberculosis patients (n = 50). Median and interquartile range is depicted, and each symbol indicates mean values of duplicates from each individual donor. Exact Mann-Whitney U test was used for comparison of groups, while paired data was evaluated by Wilcoxon Signed-Rank test. Nominal p-values are indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001.</p