J Katt

UNLABELLED: T helper (Th)17 cells are important for host defense against bacteria and fungi, but are also involved in the pathogenesis of autoimmune diseases. In primary sclerosing cholangitis (PSC), bile fluid is frequently colonized with pathogens and its strong association with inflammatory bowel disease suggests the contribution of pathogen responses to disease pathogenesis. Interleukin (IL)-17A, the signature cytokine of Th17 cells, was recently described to promote inflammation and fibrosis within the liver. Therefore, we investigated Th17 immune response to pathogens in patients with PSC. Bile fluid was obtained by endoscopic retrograde cholangiography, and bacterial and fungal species grew in the majority of samples. In addition, bacterial RNA was stained in liver sections using 16sRNA fluorescence in situ hybridization and was detected in the portal tracts in 12 of 13 tested PSC patients. Bacteria grown from patients' bile fluid were then used to stimulate peripheral blood mononuclear cells (PBMCs) and to assess their Th17 response. Compared to healthy controls or primary biliary cirrhosis patients, PBMCs from PSC patients manifested significantly higher frequencies of Th17 and Th1/Th17 cells after pathogen stimulation. The highest frequencies of Th17 cells were detected after stimulation with Candida albicans, a pathogen that has been linked to disease progression. Immunohistochemically, IL-17A-expressing lymphocytes were detected within the periductal areas of PSC patients. Th17 induction was also noted after stimulation of Toll-like receptor 5 or 7, but not of other pattern recognition receptors tested, pointing to signaling pathways potentially involved in Th17 induction in PSC. CONCLUSION: We demonstrate an increased Th17 response to microbial stimulation in patients with PSC. These data should prompt further studies investigating the link between pathogen responses, inflammation, and fibrosis in patients with PSC.

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.

The effects of GnRH pulse amplitude, frequency, and treatment duration on pituitary alpha and LH beta subunit mRNA concentrations were examined in castrate-testosterone replaced male rats. Experimental groups received iv GnRH pulses (5, 25, or 125 ng) at 7.5-, 30-, or 120-min intervals for 8, 24, or 48 h. Saline pulses were given to control rats. Acute LH secretion was measured in blood drawn before and 20 min after the last GnRH pulse. In saline controls, alpha and LH beta mRNAs (150 +/- 14, 23 +/- 2 pg cDNA bound/100 micrograms pituitary DNA) fell to 129 +/- 14 and 18 +/- 2, respectively, after 48 h. In animals receiving GnRH pulses (7.5-min intervals), the 125-ng dose stimulated a slight increase (P less than 0.01) in alpha mRNA levels after 8 and 24 h and both LH subunit mRNAs were increased by the 25- and 125-ng doses after 48 h. The 30-min pulse interval injections (25- and 125-ng doses) increased LH beta mRNA levels after 8 h, but alpha mRNAs were not elevated until after 24 h. Maximum (3-fold) increases in alpha and LH beta mRNAs were seen in rats receiving 25-ng pulses every 30 min for 48 h. Using 120-min pulses, LH subunit mRNAs were not increased by any GnRH dose through 48 h. Acute LH release was not seen in rats receiving 5 ng GnRH pulses at any pulse interval.(ABSTRACT TRUNCATED AT 250 WORDS)

EinleitungDaten aus genetischen Assoziationsstudien legen nahe, dass eine Störung des adaptiven Immunsystems an der Pathogenese der Primär Sklerosierenden Cholangitis (PSC) beteiligt ist. Wir untersuchten ob bei der PSC das Gleichgewicht zwischen Immunregulation und Inflammation gestört ist. Hierzu wurden die Frequenz und Funktionalität regulatorischer T Zellen (Treg) auf der einen Seite und die Th17 Differenzierung auf mikrobielle Stimuli auf der anderen Seite untersucht.MethodenTreg Frequenzen im peripheren Blut wurden durchflusszytometrisch bestimmt und Treg als CD4+CD25highFoxp3+CD127low Zellen definiert. Zusätzlich wurde der Anteil demethylierter DNA im peripheren Blut bestimmt. Um die Funktionalität der peripheren Tregs von PSC Patienten zu überprüfen, wurde in vitro die suppressive Kapazität von Tregs mittels CFSE Assay untersucht. Ausserdem wurden PBMCs von PSC Patienten mit hitzeinaktivierten Bakterien bzw. Hefen (E.coli, E.faecalis, S.aureus, C.albicans) stimuliert und durchflusszytometrisch analysiert.ErgebnisseIm peripheren Blut zeigte sich für PSC Patienten eine signifikant geringere Frequenz von Tregs sowohl im Vergleich zur gesunden Kontrollgruppe (p=0,0025) als auch im Vergleich zu Patienten mit Primär Biliärer Zirrhose (PBC) (p=0,0011). Im Vergleich zu Gesunden war die suppressive Kapazität der Tregs von PSC Patienten signifikant vermindert (p=0,019). Auf der anderen Seite zeigten CD4+ T-Zellen von PSC Patienten nach Stimulation mit E.faecalis (p=0,0006), nach Stimulation mit S.aureus (p=0,0051) und nach Stimulation mit C.albicans (p=0,0054) eine verstärkte Expression von IL–17 im Vergleich zu gesunden Kontrollen und PBC Patienten.SchlussfolgerungUnsere Daten weisen darauf hin, dass bei PSC Patienten ein gestörtes Gleichgewicht zwischen proinflammatorischen und regulatorischen Lymphozyten vorliegt und unterstützen daher die kürzlich beschriebenen genetischen Assoziationen bei Patienten mit PSC.

Hintergrund: IL17 spielt für die Abwehr von Krankheitserregern, aber auch für die Pathogenese verschiedener Autoimmunerkrankungen eine wichtige Rolle. Eine gestörte mucosale Barrierefunktion und fehlgeleitete Reaktion auf bakterielle Antigene, wie sie bereits für entzündliche Darmerkrankungen beschrieben wurde, könnte auch in der Pathogenese der PSC eine Rolle spielen. Wir untersuchten daher die Th17-Antwort auf mikrobielle Stimuli und TLR-Liganden in PBMCs von Patienten mit PSC, PBC und Gesunden.