JAK inhibitors disrupt T cell-induced proinflammatory macrophage activation

Objectives Macrophage subsets, activated by T cells, are increasingly recognised to play a central role in rheumatoid arthritis (RA) pathogenesis. Janus kinase (JAK) inhibitors have proven beneficial clinical effects in RA. In this study, we investigated the effect of JAK inhibitors on the generation of cytokine-activated T (Tck) cells and the production of cytokines and chemokines induced by Tck cell/macrophage interactions. Methods CD14+ monocytes and CD4+ T cells were purified from peripheral blood mononuclear cells from buffy coats of healthy donors. As representative JAK inhibitors, tofacitinib or ruxolitinib were added during Tck cell differentiation. Previously validated protocols were used to generate macrophages and Tck cells from monocytes and CD4+ T cells, respectively. Cytokine and chemokine including TNF, IL-6, IL-15, IL-RA, IL-10, MIP1α, MIP1β and IP10 were measured by ELISA. Results JAK inhibitors prevented cytokine-induced maturation of Tck cells and decreased the production of proinflammatory cytokines TNF, IL-6, IL-15, IL-1RA and the chemokines IL-10, MIP1α, MIP1β, IP10 by Tck cell-activated macrophages in vitro (p<0.05). Conclusions Our findings show that JAK inhibition disrupts T cell-induced macrophage activation and reduces downstream proinflammatory cytokine and chemokine responses, suggesting that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors.


INTRODUCTION
The hallmarks of synovitis in rheumatoid arthritis (RA) include the infiltration of inflammatory cells, predominantly bloodderived lymphoid and myeloid cells. 1 Infiltrating T-cells and monocytes/macrophages reside collaboratively within the inflamed RA synovium; hence, the established heterogeneity of inflammatory synovial macrophages 2 can drive synovitis via cytokine production by activated T-cells. 3 Conversely, T-cells activate macrophages, leading to a proinflammatory cytokine forward feedback loop. 4 Perpetuation of inflammation by monocytes/macrophages stimulated by cytokine-activated T (Tck) cells may be modelled in vitro by the macrophage: T-cell coculture assay. 5 6 Signalling pathways elicited by Tck cells in responding monocytes were identical to those induced by T-cells isolated from RA synovial tissue. 6 7 Thus, Tck cells have been proposed as a model for RA synovial T-cell-mediated stimulation of monocytes/ macrophages via cell-cell contact-dependent mechanisms. 5 7 Janus kinases (JAKs) are tyrosine kinases that recruit and activate signal transducers and activators of transcription, which drive proinflammatory cellular responses. There are four JAKs in humans, JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) 8 that subserve the biology of a range of cytokines critical in the pathogenesis of RA. 9 The JAK inhibitor tofacitinib interacts with multiple JAKs, although preferentially inhibiting JAK1dependent and JAK3-dependent cytokines. 10 WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒ Janus kinase (JAK) inhibitors modulate the activity of proinflammatory cytokines implicated in rheumatoid arthritis (RA progression.

WHAT THIS STUDY ADDS
⇒ JAK inhibition disrupts cytokine-activated T cellinduced macrophage activation, an in vitro model for RA synovial T-cell-mediated stimulation of monocytes/macrophages, and reduces proinflammatory cytokine and chemokine responses. ⇒ Suppression of the T cell: macrophage interaction may contribute to the therapeutic efficacy of JAK inhibitors.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
⇒ Although this study may not directly change policy or practice, it highlights that JAK inhibition may affect multiple steps involved in mediating inflammation by inhibiting cytokine and chemokine production and disrupting the function of innate and acquired immune cells.

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At the pathophysiological level, tofacitinib and now other JAK inhibitors, modulate the activity of proinflammatory cytokines implicated in RA progression. 10 11 Though the biological roles of JAK inhibitors in lymphocytes are well known, their function in the context of macrophage: T-cell interactions has not been defined. Here, we investigated the effect of tofacitinib, ruxolitinib and Tyrphostin AG-490 on the generation of Tck cells and assessed whether JAK inhibition in Tck cells impacts cytokine and chemokine production by macrophages in the vitro coculture system.

MATERIALS AND METHODS Isolation of peripheral blood mononuclear cells from buffy coats
Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats using Histopaque 1077 (Sigma) density centrifugation. The buffy coats were obtained from healthy volunteers from the Scottish National Blood Transfusion Service UK (SNBTS). All subjects provided informed consent with the appropriate ethical approvals in place.
Purification of CD14 + monocytes and differentiation into macrophages CD14 + monocytes were purified by positive magnetic separation using CD14 immunomagnetic microbeads (Miltenyi Biotec). Purities of >98% were routinely confirmed by flow cytometry (data not shown). CD14 + monocytes were cultured in 24 well-plates with MCSF (50 ng/mL) for 3 days. Cells were detached using a non-enzymatic dissociation solution (Sigma), washed in Dulbecco's phosphate-buffered saline and then replated in 96 well plates at a density of 5×10 5 cells/mL with MCSF (50 ng/mL) for the additional 3 days before harvesting and used in subsequent experiments.
Purification and differentiation of CD4 + Tck cells and the effect of JAK inhibition on Tck cell maturation CD4 + T-cells were isolated from PBMC using CD4 microbeads (Miltenyi Biotec). To generate Tck cells, CD4 + T-cells were cultured at 1×10 6 cells/mL with IL-2 (25 ng/mL), IL-6 (100 ng/mL) and TNF (25 ng/mL) (Peprotech) for 6 days. 12 In separate experiments, the JAK inhibitor tofacitinib was added to the culture medium during the 6-day maturation period to assess the effect of tofacitinib on Tck cell maturation. The cells were washed and cultured with mature macrophages at a 4:1 Tck: macrophage ratio. The supernatants were collected and assessed for TNF using ELISA.
Macrophages and Tck cells were also cultured alone. A 1:4 macrophage/Tck cell ratio was optimal and was used to assess the effect of JAK inhibitors. DMSO (0.001%) was used as a control. The cocultured cells were incubated for 24 hours, and supernatants were collected for analysis using ELISA or Luminex. Trans-well inserts (0.4 µm pore size; Corning) were used to assess cell-cell contact independent mechanisms.

Statistical analysis
Results were analysed using GraphPad Prism V.9 Software, (San Diego, California, USA). Statistical differences between groups were analysed using the Wilcoxon rank test, Student's t-test or one-way analysis of variance with Bonferroni post hoc corrections as appropriate.

RESULTS
Tck induce contact-dependent and concentration-dependent TNF production by human macrophages To delineate the effect of JAK inhibition on macrophages, we revisited the generation of Tck cells and their ability to activate monocyte-derived macrophages. In coculture, we established a 1:4 macrophage/Tck cell ratio as the optimal ratio of macrophages to Tck cells. In agreement with previous reports, 12 13 Tck cells activated macrophages contact-dependently, secreting TNF (online supplemental figure 1A,B).
JAK inhibitors prevent cytokine-induced maturation of Tck cells and inhibit the production of IL-6, IL-15 and IL-1RA by TcK-activated macrophages The effect of tofacitinib or ruxolitinib was evaluated on Tck cell maturation. JAK inhibitors diminished the capacity to induce TNF production by macrophages in coculture, suggesting that Tck cells failed to mature sufficiently to be able to activate macrophages ( figure 1A). The reduced TNF production was not due to cell death caused by the toxicity of DMSO ( figure 1B). Tofacitinib was selected as it is in clinical use, while ruxolitinib and AG-490 tyrphostin extended the generability of the findings. We chose a range of concentrations (online supplemental figure 2A,B) corresponding to approximate blood levels reflected by peak and trough dosage of tofacitinib by mouth. 14 We also explored the effect of JAK inhibitors

DISCUSSION
We show that tofacitinib and ruxolitinib, two structurally unrelated small molecule inhibitors, inhibit the production of TNF, IL-6, IL-15, IL-1RA and chemokines MIP1α, MIP1β, MIG and IP10 by macrophages interacting with cytokine activated memory T-cells. This was due to tofacitinib and ruxolitinib preventing cytokine-induced RMD Open RMD Open RMD Open maturation of Tck cells. Thus, tofacitinib and ruxolitinib impact the production of proinflammatory cytokines and chemokines crucial in the pathogenesis of immunemediated inflammatory diseases. Although the effects of JAK inhibition on macrophages and T-cells have been studied, [15][16][17][18] no study has thus far demonstrated the impact of JAK inhibition on Tck cell-activated macrophages.
Our observations concur with studies showing that JAK inhibitors mediate the suppression of TNF and IL-6 release, in keeping with the suggestion that JAK inhibitors work, in part, by suppressing the TNF-IFNβ-JAK-STAT1 autocrine loop. 17 Tofacitinib, ruxolitinib and AG490 Tyrphostin also led to the inhibition of Tck cell maturation. Likely, the inhibition of IL-6 and IL-15 by tofacitinib and ruxolitinib contributed to the failure to generate mature Tck cells since these cytokines are crucial in forming mature Tck cells, as previously suggested. 12 13 Indeed, we previously demonstrated that IL-15 (which signals via JAK1 and JAK3) is sufficient to form Tck cells and alters the balance between effector and memory T cells. 13 These observations suggest that JAK inhibition may effectively disrupt cell contact-mediated inflammation at the synovial site and prevent a priori development of Tck cells.
Since Tck cells in our setting were not fixed, it is likely that a small proportion of the cytokines came from Tck cells and not all from macrophages. However, using fixed Tck cells, a previous study demonstrated that Tck cellactivated macrophages induced TNF 19 ; therefore, it is reasonable to suggest that the TNF synthesis seen in our study is of macrophage origin.
The limitation of this study is that the investigation did not include RA patient samples, which would provide direct clinical relevance. However, the data suggest that JAK inhibition may effectively treat established inflammation by disrupting the crosstalk between macrophages and T-cells (online supplemental figure  3). Overall, JAK inhibition may affect multiple steps in mediating inflammation by targeting cytokine and chemokine production and affecting the function of innate and acquired immune cells, contributing to the beneficial effect of JAK inhibitors in inflammatory diseases like RA.

Treatments Treatments Treatments
Contributors MHN analysed the data and wrote the manuscript. JSN performed the assays. MF-G, DSG, SS, DP, CSG and IBM supervised the study and edited the manuscript. All authors approved the final version of the manuscript. Competing interests DP has received consultancy and sponsorship from Abbvie and Eli Lilly to attend scientific meetings. SS has received institutional research grants from Amgen (previously Celgene), Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, GlaxoSmithKline (GSK), Janssen, and UCB; and honoraria/speaker fees from AbbVie, Amgen, Eli Lilly, GSK, Janssen, and UCB. IBM has received consultancy and research support from Bristol Myers Squibb, Pfizer, Abbvie, Eli Lilly and Gilead, all of whom manufacture inhibitors of JAKs.
Patient consent for publication Not applicable.
Ethics approval This study complied with the World Medical Association Declaration of Helsinki and was approved by the Glasgow East ethics committee. Participants gave informed consent to participate in the study before taking part.
Provenance and peer review Not commissioned; externally peer reviewed.
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