Background/Purpose: Monocytes are bridging natural and acquired immunity. Information about JAK signaling in monocytes is scarce especially in an inflammatory milieu. 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 in vivo, 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 such pro-inflammatory pathways and still allow anti-inflammatory functions in monocytes.

Methods: Primary monocytes from healthy human donors were isolated and phenotyped by FACS after inhibition with JAK-inhibitor Tofacitinib and subsequent treatment with GM-CSF. Monocytes were co-cultured with autologous naïve T-cells and differentiation of Foxp3⁺ regulatory T-cells was evaluated. JAK1 activation (represented by IFNγ induced phospho-STAT1), JAK2 activation (represented by GM-CSF induced phospho-STAT5), and JAK3 activation (represented by IL-4 induced phospho-STAT6) was analyzed. Non-toxic dosages of 1-1000 nM of tofacitnib were used.

Results: We aimed to find the dose of JAK1 and JAK3 inhibition that keeps JAK2 activity (GM-CSF induced pSTAT5) intact. At 10 – 100 nM tofacitinib we detected GM-CSF-induced phospho-STAT5 while IFNγ induced phospho-STAT1 and IL-4 induced phospho-STAT6 were blocked. Phenotypic analysis showed inhibition of GM-CSF induced CD39-, CD206-, and CD209 expression with intact IL-10 expression and inhibited TNFα expression above 100 nM tofacitinib. Co-culture of GMaM and T-cells resulted in increased differentiation of Foxp3⁺ Treg that was even enhanced when 10 nM tofacitinib was used, indicating GM-CSF-signaling through STAT5 is still intact, while JAK1 and JAK 3 are inhibited in monocytes simultaneously.

Conclusion: In summary, tofacitinib (10-100 nM) facilitates GM-CSF-induced reprogramming of monocytes to anti-inflammatory cells. Pro-inflammatory activation does depend on complex interplay of multiple factors and blocking JAK/STAT activation by tofacitinib can restore the GMaM phenotype by blocking pro-inflammatory pathways while still allowing anti-inflammatory signaling in monocytes.