Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Role for IL-23 in Differentiation of Human Th17 Cells.

Kato1,  Hiroshi, Cooney2,  Laura, Endres2,  Judith, Fox3,  David A.

University of Michigan, Northville, MI
University of Michigan, Ann Arbor, MI
University of Michigan Medical Center, Ann Arbor, MI

Purpose:

Th17 cells, which are crucial in host defense, play significant roles in many autoimmune diseases, including RA. In mice, the combination of TGF-beta and IL-6 induces Th17 cells from naive CD4T cells. Whereas TGF-beta was shown to also be essential for human Th17 differentiation, it is unclear what additional pro-inflammatory cytokines are required to induce human Th17 cells. We proposed that IL-23 was dispensable for human Th17 cell differentiation given the expression of IL-23 receptor only after induction of ROR-gammat (master regulator gene of Th17 cells) in mice.

Method:

Naive CD4T cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy adult donors by a naive CD4T cell enrichment kit, and were cultured in RPMI culture media with 10%FCS, 1% Penicillin/Streptomycin, and 2% L-glutamine, with beads coated with anti-CD3/CD28 antibodies, anti-IFN-gamma and IL-4 antibodies (5ug/ml), TGF-beta (0.1, 1 or 10ng/ml), IL-1 (5ng/ml), IL-6 (10ng/ml), and IL-23 (0, 1 or 10ng/ml). Cells were collected on day 5 and incubated for 6h with phorbol ester and ionomycin followed by GolgiPlug. After blocking Fc receptors, cells were fixed, permeabilized in PBS containing 0.5% Saponin, and stained with Phycoerythrin conjugated antibodies against IL-17A, IL-17F, IL-21, or IL-22. IL-17A in culture supernatants was measured by ELISA. On day 3, total cellular RNA was extracted to synthesize cDNA. Expression of ROR-c was assessed by RT-Q-PCR with GAPDH as a reference gene.

Results:

Analysis of ELISA data from healthy subjects (n = 5) showed that IL-17A induction was more IL-23 dependent in lower TGF-beta: 0.1ng/ml; 162.8+/-70.2pg/ml (no IL-23), 233.9+/-133.0pg/ml (IL-23: 1ng/ml), 377.4+/-249.6pg/ml (IL-23: 10ng/ml) than in higher TGF-beta: 1ng/ml; 245.0 +/-115.4pg/ml (no IL-23), 338.1+/-168.0pg/ml (IL-23: 1ng/ml), 320.6+/-155.2pg/ml (IL-23: 10ng/ml). ROR-c expression was IL-23 dependent at TGF-beta concentrations of either 0.1ng/ml or 1ng/ml, but was IL-23 independent in 10ng/ml TGF-beta. These data indicate that the role of IL-23 in contributing to human Th17 differentiation varies depending on the concentration of TGF-beta. On flow cytometry, induction of IL-17A+ and IL-22+ cells was relatively independent of the concentration of IL-23. However, induction of IL-17F+ cells was more IL-23 dependent in lower TGF-beta: 0.1ng/ml whereas induction of IL-21+ cells was more IL-23 dependent in higher TGF-beta: 1ng/ml.

Conclusion:

Contrary to initial predictions, our data suggest that IL-23 might promote human Th17 differentiation either through distinct mechanisms that are in part independent of TGF-beta or through contribution to low TGF-beta driven pathway. Furthermore, each Th17-derived cytokine may be differentially regulated by IL-23. The level of secretion of Th17 cytokines is not entirely predictable by enumeration of Th17 cells that contain a specific cytokine intracellularly.

To cite this abstract, please use the following information:
Kato, Hiroshi, Cooney, Laura, Endres, Judith, Fox, David A.; Role for IL-23 in Differentiation of Human Th17 Cells. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1271
DOI: 10.1002/art.29037

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