Fingolimod Alters Tissue Distribution and Cytokine Production of Human and Murine Innate Lymphoid Cells

Abstract

Sphingosine-1 phosphate receptor 1 (S1PR1) is expressed by lymphocytes and regulates their egress from secondary lymphoid organs. Innate lymphoid cell (ILC) family has been expanded with the discovery of group 1, 2 and 3 ILCs, namely ILC1, ILC2 and ILC3. ILC3 and ILC1 have remarkable similarity to CD4+ helper T cell lineage members Th17 and Th1, respectively, which are important in the pathology of multiple sclerosis (MS). Whether human ILC subsets express S1PR1 or respond to its ligands have not been studied. In this study, we used peripheral blood/cord blood and tonsil lymphocytes as a source of human ILCs. We show that human ILCs express S1PR1 mRNA and protein and migrate toward S1P receptor ligands. Comparison of peripheral blood ILC numbers between fingolimod-receiving and treatment-free MS patients revealed that, in vivo, ILCs respond to fingolimod, an S1PR1 agonist, resulting in ILC-penia in circulation. Similarly, murine ILCs responded to fingolimod by exiting blood and accumulating in the secondary lymph nodes. Importantly, ex vivo exposure of ILC3 and ILC1 to fingolimod or SEW2871, another S1PR1 antagonist, reduced production of ILC3- and ILC1- associated cytokines GM-CSF, IL-22, IL-17, and IFN-γ, respectively. Surprisingly, despite reduced number of lamina propria-resident ILC3s in the long-term fingolimod-treated mice, ILC3-associated IL-22, IL-17A, GM-CSF and antimicrobial peptides were high in the gut compared to controls, suggesting that its long term use may not compromise mucosal barrier function. To our knowledge, this is the first study to investigate the impact of fingolimod on human ILC subsets in vivo and ex vivo, and provides insight into the impact of long term fingolimod use on ILC populations.

Keywords: FTY720; Fingolimod; ILC1; ILC3; S1PR1; SEW2871; multiple sclerosis.

Figure 1

Human ILC subsets express S1P receptors and migrate toward S1P analogs in vitro. (A) Gating strategy for human ILC subset isolation from tonsil and cord blood. (B) Sorted ILC subsets were used to determine relative gene expression of S1P receptors (S1PR1, S1PR2, S1PR3, S1PR4, S1PR5) via real time qPCR. (C) Human tonsil ILC1 and ILC3 were stained for S1PR1 to determine protein expression, a representative histogram flow plot with isotype control. (D) Migration of ILC1 and ILC3 cultured in serum free media toward FTY720 and SEW2871 gradient. Percentage of cells migrating into lower chamber of a trans-well plate quantified. (E) Migration of ILC1 and ILC3 pretreated with serum free media, FTY720 or SEW2871 for 2 h toward FTY720. Percentage of cells migrating was charted. *p < 0.05. Experiments in (B–E) were performed three separate times.

Figure 2

Fingolimod causes ILC-penia in human peripheral blood. (A) absolute number of total or subsets of ILCs in the peripheral blood of MS patients treated with fingolimod (n = 14) or untreated control MS patients (n = 11) per 10 ml peripheral blood. Total ILCs were gated as CD127⁺CD161⁺CD3⁻Lin⁻ cells, ILC3s as cKit⁺CRTH2⁻CD127⁺ CD161⁺ CD3⁻Lin⁻, ILC2s as CRTH2⁺CD127⁺CD161⁺ CD3⁻Lin⁻, and ILC1s as cKit⁻CRTH2⁻CD127⁺ CD161⁺CD3⁻Lin⁻. (B) percentages of ILCs in “A”. *Indicates p < 0.05.

Figure 3

Fingolimod causes ILC-penia, augments lymph node ILC numbers, and decreases small intestine lamina propria ILC3 numbers in mice but does not reduce antimicrobial peptide production. (A) A representative flow plot of the percentages of total ILCs in the small intestine lamina propria blood, inguinal lymph node (LN) and spleen of mice gavage-fed with fingolimod or vehicle for 30 days. The plots show live cells gated on CD90.2+CD45+B220- which then plotted as CD3/NK1.1/CD11b vs. Rorγt (top panel) or Gata3 (bottom panel). (B) Absolute number of Rorγt+ ILC3s (top panel), of Gata3+ ILC2s (middle panel) in the steady state mice after fingolimod of vehicle-gavage feeding for 30 days. Top panel, right, shows total ILCs (CD90.2-CD45+CD3-B220-CD11b-NK1.1-CD127+) in the blood of steady state mice following 30 days of fingolimod or vehicle treatment. The bottom most panel shows absolute number or percentages of IL-23RGFP+ ILC3s in the small intestine lamina propria 2-days after anti-CD40 injection following 30 days fingolimod or vehicle treatment. Three-to-four mice per group were used. Experiment was repeated 3 times. (C) 1 cm piece of ileum from fingolimod or vehicle-injected mice (for 30 days) were cultured 48 h and the supernatants were assessed with ELISA for the production of indicated ILC3-associated cytokines. Four to five of mice per group. (D) 1 cm piece of ileum or colon from fingolimod or vehicle-injected mice for 30 days was examined for gene expression of indicated antimicrobial peptides via real-time qPCR. *Indicates p < 0.05.

Figure 4

Ex vivo exposure of human ILC3s to S1P analogs inhibits production of ILC3-associated cytokines. (A) Sorted ILC3s from tonsils (gated as Lin-CD3-CD161+CD127+ckit+ CRTH2-) were cultured in complete medium at increasing doses of FTY720 and activated with IL-2, IL-23, IL-7, and IL-1B (20 ng/ml each) for 2–3 days, and PMA (50 ng/ml) /Inonomycin (1 μg/ml) stimulated for 4 h at 37°C for intracellular staining of indicated cytokines, representative flow plots (left) and quantified bar graphs for percentages of cells producing the indicated cytokines (right). (B) ELISA was performed from supernatants of “A” and SEW2871 exposed tonsil-derived (top panel) cord blood-derived (bottom panel) ILC3 cultures for ILC3-asssociated cytokines cord-derived ILC3s. (C) NKP44 surface expression by ILC3s were examined via flow cytometry, percent and mean fluorescence intensity (MFI) was quantified after culture with FTY720 for 3 days. *Indicates p < 0.05. The experiments were performed with triplicates and repeated for 3 times.

Figure 5

Fingolimod does not alter IFN-γ production by ILC1 at low doses. (A) ILC1s sorted from tonsils (gated as Lin-CD3-CD161+CD127+ckit-CRTH2-) were cultured in charcoal stripped FBS-supplemented media containing IL-12/IL-23/IL-1B/IL-7, 20 ng each, with increasing concentrations of FTY70 for 3 days IFN-γ production was measured with intracellular staining (A, a representative plot) and quantified (B) and mRNA levels were quantified via real time qPCR (C).