IL-17A Is Increased in Humans with Primary Hyperparathyroidism and Mediates PTH-Induced Bone Loss in Mice

Abstract

Primary hyperparathyroidism (PHPT) is a common cause of bone loss that is modeled by continuous PTH (cPTH) infusion. Here we show that the inflammatory cytokine IL-17A is upregulated by PHPT in humans and cPTH in mice. In humans, IL-17A is normalized by parathyroidectomy. In mice, treatment with anti-IL-17A antibody and silencing of IL-17A receptor IL-17RA prevent cPTH-induced osteocytic and osteoblastic RANKL production and bone loss. Mechanistically, cPTH stimulates conventional T cell production of TNFα (TNF), which increases the differentiation of IL-17A-producing Th17 cells via TNF receptor 1 (TNFR1) signaling in CD4(+) cells. Moreover, cPTH enhances the sensitivity of naive CD4(+) cells to TNF via GαS/cAMP/Ca(2+) signaling. Accordingly, conditional deletion of GαS in CD4(+) cells and treatment with the calcium channel blocker diltiazem prevents Th17 cell expansion and blocks cPTH-induced bone loss. Neutralization of IL-17A and calcium channel blockers may thus represent novel therapeutic strategies for hyperparathyroidism.

Keywords: IL-17; IL-17 antibody; IL-17R; PTH; T cells; Th17 cells; bone; hyperparathyroidism.

Figure 1. Primary hyperparathyroidism increases IL-17A and RORC mRNA levels in humans

Levels (Median ± interquartile range) of IL-17A and RORC mRNAs in healthy controls (n = 57) and subjects with PHPT before (n = 20) and after parathyroidectomy (n = 20). Data were analyzed by Mann Whitney (healthy controls vs. PHPT before surgery and healthy controls vs. PHPT after surgery) and Wilcoxon matched pairs signed rank tests (PHPT vs. PHPT after surgery) as the data were not normally distributed according to the Shapiro-Wilk normality test.

Figure 2. cPTH treatment expands Th17 cells and increases L-17A production

a-c. Frequency of IL-17A producing Th17 cells in unfractionated peripheral blood (PB) cells (PBC). Panel a shows representative flow cytometric dot plots from 1 mouse per group. Panel b shows the relative frequency of CD4+IL-17A+ PBC. Data are expressed as % of CD4+ cells. Panel c shows the absolute number of CD4+IL-17A+ PBCs per sample. d. IL-17A mRNA levels in PB CD4+ cells. e. IL-17A mRNA levels in unfractionated PBC and CD4+ cell-depleted PBC. f. Relative frequency of Th17 cells in the BM. g. Absolute number of BM Th17 cells per sample. h. IL-17A mRNA levels in BM CD4+ cells. i. IL-17A protein levels in BM CD4+ cells. j. mRNA levels of the Th17 cells-inducing transcription factors RORα and RORγt in BM CD4+ cells. k. Relative frequency of Th17 cells in the spleen. l. Absolute number of Th17 cells in the spleen m. IL-17A mRNA levels in spleen CD4+ cells. Data in panels b-m are shown as mean ± SEM. n = 8 mice per group in all panels. All data passed the Shapiro-Wilk normality test and were analyzed by unpaired t-tests **=p<0.01 and ***=p<0.001 compared to the corresponding vehicle group.

Figure 3. Silencing of IL-17A or IL-17A signaling prevents the bone catabolic effect of cPTH

Effects (mean ± SEM) of cPTH on bone volume, structure and turnover in mice treated with IL-17A Ab or lacking IL-1RA. a. In vitro measurements of cortical and trabecular bone indices of volume and structure by µCT scanning in WT mice treated with vehicle or cPTH and Irrelevant Ab (Irr. Ab) or anti IL-17A Ab. n= 12 mice per group. Representative 3-dimensional µCT reconstructions of the femurs are shown above the data. b. Histomorphometric indices of bone resorption (obtained in the first 10 of the 12 mice per group enrolled in the study). The images show TRAP stained sections of the distal femur used to compute the number of OCs per mm bone surface (N.Oc/BS) and the percentage of bone surface covered by OCs (Oc.S/BS),which are indices of bone resorption. Original magnification × 40. c. Dynamic indices of bone formation. The images show calcein double-fluorescence labeling used to compute mineral apposition rate (MAR) and bone formation rate (BFR), which are indices of bone formation. Original magnification × 20. The number of OBs per mm bone surface (N.Ob/BS) and the percentage of bone surface covered by OBs (Ob.S/BS), which are static indices of formation, were measured on trichrome-stained sections. d. Serum levels of CTX and P1NP. n= 12 mice per group. e. mRNA levels of RANKL in purified osteocytes (OCYs). n= 5 mice per group. f. µCT measurements of cortical and trabecular bone volume and structure in samples from WT and IL-17RA−/− mice. n = 14 mice per group. The images are 3-dimensional reconstructions of the femurs. g. Serum levels of CTX and P1NP. n = 14 mice per group. h. mRNA levels of RANKL in purified OCYs from WT and IL-17RA−/− mice. n= 5 mice per group. All data passed the Shapiro-Wilk normality test and were analyzed by 2-Way ANOVA. * = p < 0.05, ** = p<0.01 and *** = p<0.001 compared to the corresponding vehicle treated group. # = p<0.05, and ## = p<0.01 compared to IL-17 Ab cPTH. ### = p<0.001 compared to IL-17RA−/− cPTH. a= p<0.05 compared to Irr. Ab Veh. b= p<0.05 compared to WT Veh.

Figure 4. cPTH expands Th17 cells through TNF and TNFR1 signaling

a-b. Relative frequency of Th17 cells in the spleen and BM of WT and TNF−/− mice. c. IL-17A mRNA levels in BM CD4+ cells of WT and TNF−/− mice. d,e. ROR α and ROR γ t mRNA levels in BM CD4+ cells of WT and TNF−/− mice. f. Relative frequency of Th17 cells in the BM of TCRβ−/−mice previously subjected to adoptive transfer of WT T cells, TNF−/− T cells, TNFR1−/− T cells, or TNFR2−/− T cells. g. IL-17A mRNA levels in BM CD4+ cells of TCRβ−/− mice previously subjected to adoptive transfer of WT T cells, TNF−/− T cells, TNFR1−/− T cells, or TNFR2−/− T cells. h,i. Levels of RORα and RORγt mRNA in BM CD4+ cells of TCRβ−/− mice previously subjected to adoptive transfer of WT T cells, TNF−/− T cells, TNFR1−/− T cells, or TNFR2−/− T cells. Data are expressed as the mean ± SEM. n = 8 mice per group. All data passed the Shapiro-Wilk normality test and were analyzed by 2-Way ANOVA.*=p<0.05, **=p<0.01 and ***=p<0.001 compared to the corresponding vehicle group.

Figure 5. cPTH expands Th17 cells, causes bone loss and stimulates bone resorption through activation of G α S in naïve CD4+ cells

a. cPTH increases the sensitivity to TNF of naïve CD4+ cells from WT and G α S fl/fl mice but not of those from G α S^ΔCD4,8 mice. Naïve CD4+ cells were sorted from vehicle and cPTH treated mice and cultured with TNF (10–50 ng/ml) to induce their differentiation into Th17 cells. b. TNFR1 mRNA levels in BM CD4+ cells. c. TRAF2 mRNA levels in BM CD4+ cells d. Frequency of BM Th17 cells. e-g. mCT indices of bone volume and structure. h-j. Serum levels of CTX, P1NP and osteocalcin (OCN). Data are shown as mean ± SEM. n = 5 mice per group for panels b-c. n = 16 G α S fl/fl mice per group and 21 G α S^ΔCD4,8 mice per group for panels d-j. All data passed the Shapiro-Wilk normality test and were analyzed by 2-Way ANOVA. *=p<0.05, **=p<0.01, ***=p<0.001 and ****=p<0.0001 compared to the corresponding vehicle group. # = p<0.05 compared to the G α S^ΔCD4,8 cPTH group.

Figure 6. The L-type calcium channel blocker diltiazem (DIZ) prevents the effects of cPTH

a relative frequency of BM Th17 cells, b IL-17A mRNA levels in BM CD4+ cells c-d expression of RORα and RORγt mRNA in BM CD4+ cells, e-j µCT indices of bone volume and structure. k,l. Serum levels of CTX and P1NP. Data are shown as mean ± SEM. n = 12 mice per group. All data passed the Shapiro-Wilk normality test and were analyzed by 2-Way ANOVA. *=p<0.05, **=p<0.01, ***=p<0.001 and ****=p<0.0001 compared to the corresponding vehicle group. ## = p<0.01 compared to the DIZcPTH group.