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. 2023 Jan;9(1):e002671.
doi: 10.1136/rmdopen-2022-002671.

JAK inhibitors disrupt T cell-induced proinflammatory macrophage activation

Mukanthu H Nyirenda  1   2 , Jagtar Singh Nijjar  3 , Marina Frleta-Gilchrist  3 , Derek S Gilchrist  3 , Duncan Porter  3   4 , Stefan Siebert  3 , Carl S Goodyear  3   2 , Iain B McInnes  2   5
Affiliations

JAK inhibitors disrupt T cell-induced proinflammatory macrophage activation

Mukanthu H Nyirenda et al. RMD Open. 2023 Jan.

Abstract

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.

Keywords: T-lymphocyte subsets; antirheumatic agents; arthritis, rheumatoid; chemokines; inflammation.

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Conflict of interest statement

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.

Figures

Figure 1
Figure 1
Janus kinase (JAK) inhibitors prevent cytokine-induced maturation of cytokine-activated T (Tck) cells and inhibit the production of IL-6, IL-15 and IL-1RA by activated macrophages. CD14+ monocytes (5×105 cells/mL) were differentiated into macrophages by culturing them in the presence of macrophage-colony stimulating factor (MCSF; 50 ng/mL) for 6 days in a complete medium in a 96-well plate. Purified CD4+ T cells (1×106 cells/mL) were stimulated with a cocktail of IL-2, IL-6 and TNF in the absence or presence of 0.001% DMSO (used as Vehicle), tofacitinib (1000 nM) or ruxolitinib (1000 nM). The cells were incubated for 6 days to generate Tck. The Tck populations were washed and then cocultured with monocyte-derived macrophages at a concentration of 4:1 Tck-macrophage ratio. The supernatants were harvested, and the level of TNF was measured by ELISA. (A) The comparison of TNF production between DMSO and Tofacitinib (Tofa)-treated or ruxolitinib-treated Tck. (B) The comparison of TNF production between medium and DMSO-treated Tck. In separate experiments, cytokine-generated Tck were cocultured with monocyte-derived macrophages (MФs) at 4:1 ratio in the absence or presence of 0.001% DMSO (Veh) or tofacitinib (100 nM, 300 nM and 1000 nM) or ruxolitinib (100 nM, 300 nM, 1000 nM, 50 µM). Culture supernatants were harvested, and the levels of (C, D) IL-6, (E, F) IL-15 and (G, H) IL-1RA were measured using Luminex. Data represent a mean of triplicate cultures±SE of the mean of at least five experiments. Statistically significant differences are indicated (*p< 0.05; **p<0.01; ***p<0.001). DMSO, dimethyl sulfoxide; ns, not significant; Veh, vehicle.
Figure 2
Figure 2
Janus kinase (JAK) inhibitors reduce MIP1α, MIP1β, MIG and IP10 production by activated macrophages. Cytokine-generated cytokine-activated T (Tck) cells were washed and then cocultured with monocyte-derived macrophages (MФs) at a 4:1 ratio. The cells were treated with 0.001% dimethyl sulfoxide (DMSO) (Veh) or tofacitinib (100 nM, 300 nM and 1000 nM) or ruxolitinib (100 nM, 300 nM, 1000 nM, 50 µM). In some experiments, MФs were also cultured alone. Culture supernatants were harvested, and the levels of (A, B) MIP1α; (C, D) MIP1β; (E, F) MIG; and (G, H) IP10 were measured using Luminex. Data represent a mean of triplicate cultures±SE of the mean of at least five experiments. Statistically significant differences are indicated (*p<0.05; **p<0.01; ***p <0.001; ns=not significant). Veh, vehicle.
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