P118 Balancing JAK/STAT-signaling with Tofacitinib in monocytes of healthy controls and IBD patients
Cordes A.F.*1, Lenker E.2, Weinhage T.2, Varga G.2, Foell D.2
1University Hospital Münster, Department of Gastroenterology, Münster, Germany 2University Children's Hospital Münster, Department of Pediatric Rheumatology and Immunology, Münster, Germany
Monocytes are bridging natural and acquired immunity. Information about JAK signaling in monocytes is scarce. JAK-inhibition is a promising new anti-inflammatory treatment option. However, JAK/STAT activation may be involved both in pro- and anti-inflammatory monocyte programs. We have shown that GM-CSF-activated regulatory monocytes (GMaM) induce Treg-differentiation in co-cultures with naive T-cells
Primary monocytes from healthy human donors were isolated and phenotyped by FACS after treatment with GM-CSF and JAK-inhibitor Tofacitinib. Monocytes were co-cultured with autologous naïve T-cells and Foxp3+ regulatory T-cell induction was evaluated. Primary monocytes from IBD patients with active disease were used to investigate JAK/STAT signaling and inhibition. JAK1 activation (represented by IFN Υ -induced phospho-STAT-1), JAK2 activation (represented by GM-CSF-induced phospho-STAT5)and JAK3 activation (represented by IL-4-induced phospho-STAT6) was analyzed by FACS. Non-toxic dosages of 1–1000 nM Tofacitnib were used.
We aimed to define the dose of JAK inhibition that keeps JAK2 activity (GM-CSF-induced pSTAT5) intact. At 10–100 nM Tofacitinib we found GM-CSF-induced phospho-STAT5 while phospho-STAT1 and phospho-STAT6 were still blocked. Concentrations above 100 nM Tofacitinib led to inhibition of GM-CSF-induced CD39-, CD206-, CD209-expression. 10–100 nM allowed CD39-, CD206-, CD209 expression and IL-10 release while TNFα was still blocked. Co-culture of GMaM and T-cells resulted in increased differentiation of Foxp3+ Treg that was even enhanced when 10nM Tofacitinib was used. Investigation of JAK/STAT activation in monocytes from IBD patients revealed a higher base line phosphorylation of STATs with lower increase after stimulation compared to healthy controls. Furthermore, TNFα expression was not inhibited in monocytes of IBD patients using a similar Tofacitinib dosage which led to TNFα inhibition in healthy controls (10–100 nM).
In summary, Tofacitinib (10–100 nM) facilitates GM-CSF-induced reprogramming of monocytes to anti-inflammatory cells. This could not be confirmed in monocytes from active IBD patients as blockage with similar doses did not inhibit pro-inflammatory cytokine expression. Thus, pro-inflammatory activation in IBD does depend on more complex interplays of multiple factors and solely blocking JAK/STAT activation by Tofacitinib cannot fully restore the GMaM phenotype.