Background: 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 in vitro. Inflammatory T-cells produce high amounts of GM-CSF, not leading to anti-inflammatory monocytes, likely because of pro-inflammatory cytokines in the environment. We used JAK-inhibitor Tofacitinib to explore mechanisms that block pro-inflammatory pathways and allow anti-inflammatory functions in monocytes.
Methods: 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.
Results: 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).
Conclusions: 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.