The retinoic acid receptor-α modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression

Abstract

Background: Th2 cytokine responses are enhanced by all trans retinoic acid (ATRA), the bioavailable form of vitamin A. Retinoic acid receptor alpha (RARα) is the high affinity receptor for ATRA that mediates these pro-Th2 effects. We have previously characterized two major human Th2 subpopulations: IL-5- Th2 (IL-5-, IL-4+, IL-13+) and IL-5+ Th2 cells (IL-5+, IL-4+, IL-13+), which represent less and more highly differentiated Th2 cells, respectively. We hypothesized that the pro-Th2 effects of ATRA may differentially affect these Th2 subpopulations.

Methods: Specific cytokine producing Th2 subpopulations were identified using intracellular cytokine staining. Proliferation was measured using the Cell Trace Violet proliferation tracking dye. Apoptotic cells were identified using either annexin-V or active caspase 3 staining. Th2 gene expression was measured using quantitative polymerase chain reaction.

Results: ATRA increased the output of Th2 cells from house dust mite allergen (HDM) specific short-term cell lines, and this enhancement was limited to the IL-5+ Th2 subpopulation. Conversely, the RARα antagonist Ro415253 decreased Th2 cell output from these cultures, and this effect was again limited to the IL-5+ Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited Th2 cell proliferation, and this affect was more pronounced for the IL-5+ vs. IL-5- Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited the expression of IL5 in a significant manner, which was not found for IL4 or IL13.

Conclusions: We report that the reciprocal regulation of Th2 cytokine expression and proliferation by RARα modulators are largely limited to modulation of IL-5 gene expression and to proliferation of the highly differentiated IL-5+ Th2 subpopulation. These results suggest that RARα antagonism is a potential means to therapeutically target allergic inflammation.

Trial registration: Clinicaltrials.gov identifier: NCT01212016.

Figure 1

Enhancement of Th2 cell output in HDM-stimulated PBMC cultures is reciprocally regulated by RARα modulators. Cell trace violet (CTV)-labeled PBMCs from allergic asthmatic subjects were stimulated with HDM Ag extract (40 U/ml) for 10d in the presence of ATRA (RARα agonist), Ro41 (RARα antagonist), or DMSO vehicle control. After 10d cultures were restimulated with PMA and ionomycin and ICCS was performed. (A) Representative flow plots are shown. Combined results from 3 cultures showing (B) the frequency of CTV^low cells, after gating on viable HDM proliferated CD3+, CD4+, CD8- cells. Combined results from 3 cultures showing (C) the cell number and (D) percentage of Th2 cytokine producing HDM-expanded T cells. (E) Representative flow plots showing IL-13 vs. IL-5 expression after gating on viable HDM proliferated CD3⁺ CD4⁺ CTV^low cells. Combined results from 3 cultures showing (F) cell number and (G) percentage of of IL-5⁺ (IL-5⁺ IL-13⁺) and IL-5^- (IL-5^- IL-13⁺) Th2 cell populations. Data points represent independent HDM-stimulated PBMC cultures. P-values were generated with 2-way ANOVA. In B and C, only 2 experiments were performed using IL-4 as an analyte.

Figure 2

RARα modulators intrinsically regulate IL-5+ Th2 cell proliferation. In vitro differentiated Th2 cells were CTV labeled and activated with the noted stimuli in the presence and absence of ATRA or Ro41. After 8d cells were restimulated with PMA and ionomycin and ICCS was performed. (A) Representative flow plot showing total IL-5 vs. CTV (top panel), as well as CTV staining of IL-5+ Th2 (middle panel), and IL-5- Th2 (bottom panel) subpopulations. Combined results from 5 Th2 cultures showing CTV^low percentages of (B) total, (C) IL-5⁺ Th2, and (D) IL-5^- Th2 subpopulations. Data are presented with means (bar) and p values were generated via paired Student’s t test.

Figure 3

ATRA does not affect apoptosis of HDM-expanded Th2 cells. CTV labeled PBMCs from allergic asthmatic subjects were cultured with HDM Ag for 7d with ATRA or DMSO vehicle control. Annexin V staining was measured in the CTV^low (unprolferated) and CTV^high (proliferated HDM Ag specific) cells, after first gating on CD4+, 7AAD- lymphocytes. Above, data from a representative experiment. Below, combined results from 3 independent experiments. The percentage and number of annexin V positive cells are plotted. No statistical differences were observed by Student’s t test.

Figure 4

ATRA does not affect caspase-3 activation in CD3 stimulated Th2 cells. Th1 and Th2 cell lines were generated in vitro then activated with anti-CD3 antibody in presence of ATRA or DMSO vehicle control. Cultures were harvested 4d later and stained for activated caspase-3. 2xTh2, 3xTh2, etc. represent 2, and 3 serial 7d cycles of Th2 differentiation. Above, flow cytometry plots are shown for all cell lines. Below, combined results from all cultures noting the percentage of viable (Live/Dead negative) activated caspase-3+ cells (mean +/- SEM). No statistical differences were observed by paired Student’s t test.

Figure 5

ATRA inhibits in vitro Th2 cell differentiation. Naïve CD4 T cells were isolated by MACS and differentiated under Th2 culture conditions in the presence of 1nM ATRA or DMSO vehicle control. (A, B) After 2 and 3 rounds of differentiation (2xTh2 and 3xTh2, respectively) cells were restimulated with PMA and ionomycin and analyzed by ICCS. (C, D) Percentages and (E, F) mean fluorescence intensity (MFI) of cytokine positive 2xTh2 (C, E) and 3xTh2 (D, F) cells are plotted from 3 experiments. Data is presented as mean +/- SEM. 2-way ANOVA was performed and Bonferroni multiple comparisons p values are displayed.

Figure 6

ATRA and Ro41 reciprocally regulate IL5 gene expression.In vitro differentiated 2xTh2 (open symbols) and 3xTh2 (closed symbols) cells were cultured with ATRA, Ro41, or DMSO vehicle for 6hrs. The expression of IL-5, IL-4, and IL-13 were determined by qRT-PCR and fold change (treated/vehicle) plotted. Th2 cell lines stimulated in the presence of IL-2 (50 U/ml) only. Data points represent individual Th2 cell lines. Paired Student’s t test was used to evaluate the data. Each symbol represents a specific cell line.

Figure 7

The human IL-5 promoter (0 to -1054) contains a putative retinoic acid response element (RARE). A RARE contains two RARE ½ sites [G(T/G)TCA] and from 1 to 5 spacer nucleotides designated by the “DR” designation. (A) A Schematic representation of the IL-5 locus between species. The putative RARE location in the IL5p is compared between human, mouse, rat, and rhesus. Alignment of DNA sequence around the IL5p putative RARE between (B) human and rhesus, and (C) mouse and rat.