Background/Purpose:

T helper-type 17 (Th17) cells are proinflammatory CD4+ cells characterized by the production of Interleukin-17 (IL-17).There is evidence that IL-17 and other cytokines which Th17s produce such as IL-21 are involved in the pathogenesis of RA.¹ Lipopolysaccharide (LPS)-stimulated monocytes can promote differentiation of CD4+ cells into Th17 cells and produce IL-17 in vitro.² This study examined the effect of 4 anti–tumor necrosis factor (TNF) agents (adalimumab, etanercept, infliximab, and certolizumab pegol) on the expansion of CD4+CD45RO+ memory T cells into Th17 cells driven by LPS-stimulated monocytes.

Methods:

Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers. Monocytes and CD4 + T cells were purified from the PBMC by positive and negative selection, respectively. CD4+CD45RO+ memory T cells were enriched from the CD4+ T cell fraction by positive selection. Purified monocytes and memory T cells were co-cultured at a 1:1 ratio for 7 days with CD3/CD28 Human T-Activator Dynabeads with and without 1 μg/mL LPS. Cells were cultured in the presence and absence of the 4 anti-TNF agents at 10 μg/mL. After 7 days the CD4+ T cells were stained for intracellular Interferon γ (IFNγ) and IL-17A and analyzed by flow cytometry. IL-17A and IL-17F secretion into the supernatant was determined by ELISA.

Results:

CD4+ T cells positive for IL-17A were increased from 5.7% in the control co-cultures without LPS to 20.5% with LPS (mean of 2 experiments). The frequency of IFNγ-positive CD4+ T cells showed a smaller increase from 4.4% to 10.6% when LPS was added. IL-17A and IFN-γ were expressed largely by different cells, suggesting the expansion of both Th17 and Th1 T-helper subsets. The frequency of IL-17A-producing CD4+ T cells in co-cultures of monocytes and memory T cells plus LPS in the presence of the 4 anti-TNF agents were roughly 2.5 -fold lower than the LPS-positive control cultures generated in the absence of anti-TNF agents (mean of 4 experiments). Cells exposed to the 4 anti-TNF agents showed a similar level of CD4+ cells producing IL-17A and IFNγ. The level of IL-17A secreted into the supernatant decreased from 580 pg/mL in the LPS positive control to 180 pg/mL in co-cultures generated in the presence of the 4 anti-TNF agents. IL-17F decreased from approximately 8 ng/mL to 2 ng/mL in the LPS control and the anti-TNF exposed cultures, respectively (mean of 4 experiments). There were no significant differences in the concentration of IL-17A or IL-17F from co-cultures exposed to the 4 different anti-TNF agents.

Conclusion:  

The increased frequency of IL-17+ T cells and secretion of IL-17A and IL-17F suggest that LPS-activated monocytes support the expansion of Th17 cells present within the memory pool. Exposure to anti-TNF agents inhibited Th17 expansion and IL-17A production. This suggests that part of the mode of action of anti-TNF agents may be to reduce Th17 expansion and, as a consequence, IL-17A and IL-17F concentration. It is unclear whether soluble TNF or membrane TNF is responsible for this activity.

References:

1. Pernis AB. J Intern Med. 2009;265:644–652.

2. Evans H, et al. Proc Natl Acad Sci U S A. 2009;106:6232‒6237.