Divergent Effect of Tacalcitol (PRI-2191) on Th17 Cells in 4T1 Tumor Bearing Young and Old Ovariectomized Mice

Abstract

Vitamin D and its analogs are known for their role in the development of breast cancer and in immunomodulation. Our previous studies have shown the pro-metastatic effect of calcitriol and tacalcitol (PRI-2191) in young mice bearing 4T1 breast cancer and the anti-metastatic effect in aged ovariectomized (OVX) mice. Therefore, the aim of our work was to characterize Th17 cell population in young and aged OVX mice bearing 4T1 tumors treated with calcitriol and PRI-2191. The expression of genes typical for Th17 cells was examined in splenocytes, as well as splenocytes differentiated with IL-6 and TGF-β to Th17 cells (iTh17). Expression of genes encoding vitamin D receptor (Vdr) and osteopontin (Spp1) as well as the secretion of IL-17A were evaluated in iTh17 cells. PRI-2191 treatment increased the expression of Rora and Rorc transcription factors, Il17a, Il17re and Il21 in iTh17 cells from young mice. In aged OVX mice this effect was not observed. Increased expression was observed in the case of Vdr and Spp1 genes in iTh17 cells from young mice treated with PRI-2191. What is more, in young mice treated with PRI-2191 the secretion of IL-17A to the culture media by iTh17 cells was increased, whereas in aged OVX mice a significant decrease was noted. Increased expression of Spp1 in young mice treated with PRI-2191 may enhance the differentiation of Th17 cells.

Keywords: IL-17A; PRI-2191; Th17; breast cancer; calcitriol; metastasis.

Figure 1.

The expression of selected Th17 genes in splenocytes of young (A) and aged (B) mice and in lymph nodes of young mice (C) bearing 4T1 mammary gland tumors. 50 ng of cDNA from untreated splenocytes and 30 ng from lymph nodes were used for a single real-time PCR reaction. Each sample was performed in a triplicate in a single experiment. The real-time PCR reaction was carried out in the Viia 7 device equipped with Viia 7 Software v1.1 software. PCR amplification cycles were performed at 2 min 50ºC, 10 min 95ºC, and then 50 cycles: 95ºC 10 sec, 58ºC 45 sec using specific primers with TaqMan chemistry. Fold-change (RQ) of target cDNA was determined using the ΔΔCt method in reference to the beta-2 microglobulin control gene (B2m; Mm00437762_m1) in the DataAssistTM v3.0.1 software. Number of mice: 5-6/group. Data presented as mean with SD. Statistical analysis: Dunn’s multiple comparison test, *p<0.05.

Figure 2.

Characteristics of CD4+ splenocytes (NT) stimulated with IL-6 and TGF-β (iTh17). The expression of genes typical for Th17 cells in cells from young (A) and aged (B) mice (day 21). Expression of Vdr and Spp1 in iTh17 cells induced from CD4+ splenocytes from young (C) and aged OVX (D) mice. The representative diagrams illustrating the purity of isolated CD4+ splenocytes from young (E) and aged OVX (F) control mice and mice treated with calcitriol. Panel A represents non-stained cells, panel B shows cells stained with anti-CD4 antibody. For a single real-time PCR reaction diluted 1:5 cDNA from previously preamplified stimulated iTh17 splenocytes was used. Each sample was performed in triplicate in a single experiment. Fold-change (RQ) of target cDNA was determined using the ΔΔCt method in reference to the beta-2 microglobulin control gene (B2m; Mm00437762_m1) in the DataAssistTM v3.0.1 software and then calculated as a relative to the values obtained for non-stimulated splenocytes from control mice. Number of mice: 4/group with the exception of control iTh17 cells from aged mice N=3. Data presented as mean with SD. Statistical analysis: Dunn’s multiple comparison test, *p<0.05.

Figure 3.

IL-17A production by iTh17 cells differentiated from CD4+ T lymphocytes (NT) from spleens of young and aged mice bearing 4T1 mammary gland cancer. Number of mice: 5-6/group. Data presented as mean with SD and min-max values. Statistical analysis: Dunn’s multiple comparison test, *p<0.05

Figure 4.

Plasma level of 17β-estradiol in aged OVX mice bearing 4T1 tumors and treated with calcitriol or PRI-2191. Number of mice: 4-5/group. Data presented as mean with SD. Statistical analysis: Dunn’s multiple comparison test, *p<0.05.

Figure 5.

Summary of the effects on iTh17 cells of PRI-2191 (tacalcitol) treatment of young and aged OVX mice bearing 4T1 mammary gland cancer. (A) PRI-2191, by increasing osteopontin level, may indirectly potentiate the differentiation of Th17 cells and the secretion of IL-17 by them. The direct effect of PRI-2191 after binding with VDR on the expression of osteopontin and genes responsible for Th17 differentiation is also considered. (B) In old OVX mice, the observed reduction in the secretion of osteopontin may contribute to reduced secretion of IL-17. Continuous red lines show the effects of PRI-2191 observed in the orthotopic 4T1 mouse breast cancer model, where PRI-2191 stimulated metastasis in young mice and inhibited in old OVX mice. Dotted lines indicate the effects of osteopontin on Th17 cells differentiation (through CD44 or other receptors) observed by other authors in other models (non-cancerous).