Baricitinib therapy response in rheumatoid arthritis patients associates to STAT1 phosphorylation in monocytes

Abstract

Baricitinib is a Janus kinase (JAK) 1 and 2 inhibitor approved for treating rheumatoid arthritis (RA). The JAK/STAT system is essential in the intracellular signaling of different cytokines and in the activation process of the monocyte lineage. This study verifies the effects of baricitinib on STAT phosphorylation in monocytes of RA patients and evaluates the correlation between STAT phosphorylation and response to therapy. We evaluated the disease activity of patients (DAS28CRP) at baseline (T0) and after 4 and 12 weeks (T1-T3) of treatment with baricitinib, dividing them into responders (n = 7) and non-responders (n = 7) based on the reduction of DAS28CRP between T0 and T1 of at least 1.2 points. Through flow cytometry, STAT1 phosphorylation was analyzed at T0/T1/T3 in monocytes, at basal conditions and after IL2, IFNα, and IL6 stimulation. We showed that monocyte frequency decreased from T0 to T1 only in responders. Regarding the phosphorylation of STAT1, we observed a tendency for higher basal pSTAT1 in monocytes of non-responder patients and, after 4 weeks, a significant reduction of cytokine-induced pSTAT1 in monocytes of responders compared with non-responders. The single IFNα stimulation only partially recapitulated the differences in STAT1 phosphorylation between the two patient subgroups. Finally, responders showed an increased IFN signature at baseline compared with non-responders. These results may suggest that monocyte frequency and STAT1 phosphorylation in circulating monocytes could represent early markers of response to baricitinib therapy.

Keywords: ISGs; JAKi; STAT; interferon; monocytes; rheumatoid arthritis.

Figure 1

Monocyte frequency and STAT1 phosphorylation in response to a cytokine cocktail in R versus NR patients. (A) Representative overlays showing monocytes percentages in PBMCs (upper panels) and STAT1 phosphorylation (pSTAT1) in untreated (−cytokines) or cytokine cocktail-stimulated (+cytokines) monocytes (lower panels) in PBMCs of R and NR patients (n = 4/group), collected before (T0) and after 1 (T1) month post baricitinib therapy starting. Numbers in tables indicate the gMFI of pSTAT1 in each condition; fmo, fluorescence-minus-one control. (B, C) Cumulative analysis of monocyte percentage (B) and the rgMFI of pSTAT1 in monocytes (C), calculated by subtracting the gMFI of the respective fmo control, in PBMCs of R and NR patients (n = 4/group), collected before (T0) and after 1 (T1) month post baricitinib therapy starting. Bars represent means P-values were calculated by 2-way ANOVA with Geisser–Greenhouse correction and Sidak’s multiple comparisons test.

Figure 2

Monocyte frequency and subpopulations in R versus NR patients. (A, B) Representative overlays (A) and distributions (B) of monocyte percentages in PBMCs of R (n = 5) and NR (n = 6) patients, collected before (T0) and after 1 (T1) and 3 (T3) months post baricitinib therapy starting. P-values were calculated by 2-way ANOVA with Geisser–Greenhouse correction and Sidak’s multiple comparisons test. (C, D) Representative contour plots (C) and stacked histograms (D) showing the percentage of classical, intermediate, and nonclassical monocytes in PBMCs of R (n = 5) and NR (n = 6) patients at the indicated time points. P-values were calculated by 2-way ANOVA with Geisser–Greenhouse correction and Sidak’s multiple comparisons test, comparing classical monocyte frequencies in the indicated conditions.

Figure 3

STAT1 phosphorylation in monocytes of R versus NR patients. (A) Representative contour plots showing the percentages of pSTAT1+ cells in gated classical monocytes from PBMCs of one R and one NR patient, collected before (T0) and after 1 (T1) and 3 (T3) months post baricitinib therapy starting. Cells were stained after culture for 30 min with (+IFNα) or without (−IFNα) 4 ∗ 10⁴ IU/ml of IFNα. (B–D) Cumulative distributions of the percentages of pSTAT1+ cells without IFNα stimulation (B) or with IFNα stimulation (C), and of the difference (Δ) between the two values calculated for each sample and condition (D). The analysis was performed in PBMCs of R (n = 5) and NR (n = 6) patients at the indicated time points. Samples were included in the analysis only if enough cells were found in the monocyte gate. P-values were calculated by 2-way ANOVA with Geisser–Greenhouse correction and Sidak’s multiple comparisons test.

Figure 4

Expression of ISGs in monocytes at therapy starting of R versus NR patients. (A) Representative histogram overlays plots showing the expression of ISG15, PKR, and MXA by intracellular staining in gated classical monocytes from the PBMCs of one R and one NR patient collected at baricitinib therapy starting (T0). Gray histograms represent the fmo, fluorescence-minus-one control; red represents cells stained ex vivo; green represents cells stained after overnight (o.n.) culture; blue represents cells stained after o.n. culture in the presence of 4 ∗ 10⁴ IU/ml of IFNα. Numbers in tables indicate the gMFI. (B–D) Cumulative distributions of the intracellular expression (measured as the gMFI) of ISG15 (B), PKR (C), and MXA (D), in gated classical monocytes from PBMCs of R and NR patients (n = 5/group) collected at baricitinib therapy starting (T0), and stained ex vivo or after overnight (o.n.) culture in the absence or the presence (o.n. + IFNα) of 4 ∗ 10⁴ IU/ml of IFNα. The dotted lines indicate the average fmo values for each protein. P-values were calculated by Mann–Whitney test. .