P026 Differential effects of tofacitinib on macrophage activation may contribute to lack of response in ulcerative colitis patients

Abstract

Abstract Background We previously showed that ulcerative colitis (UC) patients refractory to tofacitinib present an increase in the activation and the pro-inflammatory profile of intestinal macrophages1. However, the mechanism by which tofacitinib promotes further activation of macrophages in these group of patients remains unknown. Methods Blood monocytes from healthy individuals were cultured in the presence of M-CSF as previously described2. The differentiated macrophages were treated with LPS (10ng/ml), TNF (20ng/ml) or IFNγ (5ng/ml) in the presence or absence of tofacitinib (300nM). The effect of tofacitinib on gene expression and protein production in response to each stimulus was measured by qPCR and ELISA, respectively. Single-cell RNAseq (scRNAseq) data generated in a cohort of UC patients treated with tofacitinib1 was compared to the bulk transcriptome of monocyte-derived macrophages stimulated with LPS +/- anti-IL-10 mAb3. Results Monocyte-derived macrophages treated with tofacitinib significantly reduced the expression of activation markers downstream from IFNγ or TNFα. In contrast, the response to LPS was either maintained or intensified by tofacitinib as seen by the increase in expression of inflammatory marker genes including chemokines and cytokines CXCL1, CXCL5, IL1B, TNFA and IL6, and M1-like markers INHBA and CLEC5A (Figure 1A). Protein analysis of cell supernatants confirmed these observations. Compared to IFNγ and TNF, LPS drives the production of high concentrations of the anti-inflammatory IL-10 (Figure 1B) which can act on macrophages through a JAK-dependent signaling pathway. We hypothesized that by inhibiting IL-10 autocrine signaling on LPS-activated macrophages tofacitinib would promote their hyperactivation. Indeed, the transcriptional profile of LPS-stimulated macrophages treated with a blocking anti-IL-10 antibody3 highly overlapped with those genes that were upregulated in macrophages from patients refractory to tofacitinib, including IL1B, IL6, INHBA and IDO1 (Figure 1C). This overlap was not observed in responder patients. Conclusion Based on the combination of biopsy scRNAseq from tofacitinib-treated patients and in vitro macrophage cultures, we suggest that lack of response to tofacitinib is driven by its effect on IL-10 signaling. While IL-10 is essential to control macrophage activation when stimulated with the pathogen molecular pattern LPS, it plays no significant role when macrophages are activated with IFNγ or TNF. Hence, we hypothesize that LPS-dependent inflammation may predispose to lack of response to tofacitinib.