Effect of 1,25(OH)₂D3 on the production of inflammatory cytokines by HRPTEpiC, induced by activated CD4⁺ T cells.

<p><b>(A)</b> IL-6 and <b>(B)</b> IL-8 production by HRPTEpiC which were pre-treated with 1,25(OH)₂D3 (10 nM) as indicated, and then co-cultured for 48 hours with activated CD4+T cells. Note that treatment with 1,25(OH)₂D3 did suppress the production of IL-6 and IL-8 by HRPTEpiC. *P<0.05 vs. CD4⁺ T control and ^†P<0.05 vs. activated CD4⁺ T. Values are expressed as the mean and SD of triplicate cultures. 1,25(OH)₂D3, 1α,25 dihydroxy-vitamin D3; HRPTEpiC, human renal proximal tubular epithelial cells</p

Increase of Th17 Cell Phenotype in Kidney Transplant Recipients with Chronic Allograft Dysfunction

<div><p>This study was performed to determine the association of Th17 cell phenotype with chronic allograft dysfunction in kidney transplant recipients (KTRs). We compared the expression of Th17 cell phenotype in KTRs with chronic allograft dysfunction group (CAD, n = 52) with four control groups (long-term stable KTRs (LTS, n = 67), early stable KTRs (ES, n = 28), end stage renal disease (ESRD, n = 45), and healthy control (HC, n = 26). We also performed in vitro study using human proximal renal tubular epithelial cell line (HPRTEpiC) to evaluate the effect of IL-17 on human renal tubular epithelial cells. The CAD group showed increased percentage of Th17 cells out of CD4⁺ T cells and also increased proportion of IL-17 producing cells out of effector memory T cells or out of CCR4⁺CCR6⁺/CD4⁺ T cells compared to the LTS group and other control groups. Also, the serum level of IL-17, IL-33, and RAGE, and the expression of IL-1beta, RAGE, and HMGB1 mRNA showed an increase in the CAD group compared to the LTS group. In vitro study revealed that IL-17 increased production of IL-6 and IL-8 and up-regulated profibrotic gene expression such as ACTA-2 and CTGF in HPRTEpiC in a dose-dependent manner, which suggests that IL-17 has a role in the development of renal tubular cell injury. The results of our study may suggest that increase of Th17 cell phenotype could be a marker for the chronic allograft injury; hence there is a need to develop diagnostic and therapeutic tools targeting the Th17 cells pathway.</p></div

Protective effect of 1α,25-dihydroxyvitamin D3 on effector CD4⁺ T cell induced injury in human renal proximal tubular epithelial cells

<div><p>Background</p><p>The aim of this study was to investigate the protective effect of 1α,25-dihydroxyvitamin D3 [1,25(OH)₂D3] on effector CD4⁺ T cells or on inflammatory cytokine-induced injury in human renal proximal tubular epithelial cells (HRPTEpiC).</p><p>Methods</p><p>First, we investigated the effect of 1,25(OH)₂D3 on CD4⁺ T cell proliferation. Second, we examined the effect of 1,25(OH)₂D3 on inflammatory cytokine secretion or fibrosis in HRPTEpiC induced by inflammatory cytokines or activated CD4⁺ T cells using ELISA and real-time PCR. Lastly, we compared urine inflammatory-cytokine (IL-6, IL-8) or KIM-1 levels in kidney transplant recipients low serum 25-hydroxyvitamin D (25(OH)D) group (< 20 ng/mL) (n = 40) and normal 25(OH)D group (n = 50).</p><p>Results</p><p>Pre-incubation with 1,25(OH)₂D3 significantly reduced the percentages of Th1 and Th17 cells compared to that of Th0 condition (P < 0.05 for each). In contrast, 1,25(OH)₂D3 increased the proportion of Th2 and Treg cells in a dose-dependent manner (P < 0.05 for each). Treatment of HRPTEpiC with inflammatory cytokines (TNF-α, IL-17, and TGF-β) or effector CD4⁺ T cells resulted in increased production of IL-6, IL-8, or KIM-1 from HRPTEpiC in a dose-dependent manner. However, treatment with 1,25(OH)₂D3 significantly reduced the level of these cytokines (P < 0.05 for all). Western blot analysis demonstrated that the mTOR/STAT3/ERK pathway was downregulated by 1,25(OH)₂D3 in HRPTEpiC. Furthermore, the concentrations of urine IL-6/creatinine (P < 0.05) and Kim-1/creatinine (P < 0.05) were higher in the low 25(OH)D group than in the normal 25(OH)D group in kidney transplant recipients.</p><p>Conclusion</p><p>The results of this study suggests that vitamin D may have a significant role in the regulation of inflammation in allograft tissue in kidney transplant recipients.</p><p>Trial registration</p><p>All participants provided written informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital (<a href="https://clinicaltrials.gov/ct2/show/KC13TNMI0701" target="_blank">KC13TNMI0701</a>).</p></div

Distribution of Th1, Th2, Th17 and Treg subpopulations out of CD4⁺ T lymphocytes.

<p><b>(A)</b> PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ T cells were gated for further analysis. <b>(B)</b> The proportion (%) of IFN-γ⁺/CD4⁺ T cells <b>(C)</b> IL-4⁺/CD4⁺ T cells <b>(D)</b> IL-17⁺/CD4⁺ T cells <b>(E)</b> CD25⁺FOXP3⁺/CD4⁺T cells in each patient group. * <i>P</i><0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.</p

Effect of 1,25(OH)₂D3 on the production of inflammatory cytokines by HRPTEpiC, induced by recombinant human IL-17 (rhIL-17) or human TNF-α.

<p>HRPTEpiC were cultured with rhIL-17 (0, 1, 10, 50, or 100 ng/mL) or TNF-α (0, 1, 10, or 50 ng/mL) for 48 hours, and the production of <b>(A)</b> IL-6 and <b>(B)</b> IL-8 was measured (n = 3). Note that IL-6 and IL-8 levels were significantly increased by IL-17 or TNF-α in a dose-dependent manner. *P<0.05 vs. Nil and ^†P<0.05 vs. IL-17 1 ng/mL and ^‡P<0.05 vs. TNF-α 1 ng/mL. <b>(C)</b> IL-6 and <b>(D)</b> IL-8 production by HRPTEpiC which were pretreated with 1,25(OH)₂D3 (10 nM) as indicated, and then cultured for 48 hours with IL-17 (0, 10, or 50 ng/mL) or TNF-α (10 ng/mL). Note that the addition of 1,25(OH)₂D3 significantly decreased the IL-6 or IL-8 level, which was increased by IL-17 or TNF-α. *P<0.05 vs. Nil and ^†P<0.05 vs. IL-17 50ng/mL and ^‡P<0.05 vs. IL-17 10ng/mL + TNF-α 10 ng/mL.</p

Effect of 1,25(OH)₂D3 on the production of KIM-1 and fibronectin 1 from HRPTEpiC induced by recombinant human IL-17 (rhIL-17), human TNF- α or TGF-beta.

<p><b>(A)</b> The expression of KIM-1 by HRPTEpiC was pretreated with 1,25(OH)₂D3 (10 nM) as indicated, and then cultured for 24 hours with TNF-α (50 ng/mL) and/or IL-17 (50ng/mL) (n = 3). The expression of KIM-1 was measured by real-time PCR. Note that addition of 1,25(OH)₂D3 significantly decrease KIM-1 expression which was increased by TNF-α and IL-17. *P<0.05, **P<0.01 vs. Nill and ^†P<0.05 vs. TNF- α 50 and ^‡P<0.05 vs. TNF-α+IL-17. <b>(B)</b> The expression of Fibronectin-1 by HRPTEpiC was pretreated with 1,25(OH)₂D3 (10 nM) as indicated, and then cultured for 24 hours with TGF-β (10 ng/mL) and/or IL-17 (50ng/mL) (n = 3). The expression of fibronectin-1 was measured by real-time PCR. 1,25(OH)₂D3 significantly decreased fibronectin-1 expression, which was increased by TGF-β (10 ng/mL) and IL-17. **P<0.01 vs. Nill and ^#P<0.05 vs. TGF- β 10 and ^##P<0.05 vs. TGF- β+IL-17.</p

Distribution of T_naïve, T_CM, T_EM subpopulations of CD4⁺T lymphocytes and IL-17⁺/T_EM subpopulations of CD4⁺ T lymphocytes.

<p><b>(A)</b> PBMCs were stained with anti-CD4 PE-cy7, anti-CD45RA–FITC, anti-CCR7 APC and anti-IL-17 PE. CD4+ T cells were gated for further analysis. <b>(B)</b> The proportion (%) of T_naïve/CD4⁺ T (CD45RA^<i>+</i>CCR7⁺/CD4⁺ Tcells) <b>(C)</b> T_CM/CD4⁺ T (CD45RA^–CCR7⁺/CD4⁺Tcells) <b>(D)</b> T_EM/CD4⁺ T (CD45RA^–CCR7^–/CD4⁺ Tcells) <b>(E)</b> After surface staining with CD45 and CCR7 mAbs, analysis of IL-17 in CD4⁺ T cell subsets by intracellular flow cytometry was done. <b>(F)</b> The proportion (%) of IL-17⁺/T_EM. in each patient group. * <i>P</i><0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.</p

Effects of 1,25(OH)₂D3 on the expression of mTOR and STAT3 proteins in HRPTEpiC.

<p><b>(A)</b> Immunoblotting of VDR, <i>p</i>-mTOR, mTOR, <i>p-</i>Akt, Akt, <i>p-</i>s6k, <i>p-</i>STAT3(705), STAT3, <i>p</i>-ERK, ERK and REDD1 in HRPTEpiC pretreated with or without 1,25(OH)₂D3 (10 nM) and then cultured with recombinant IL-17 for 1 hour. <b>(B)</b> Stimulation of HRPTEpiC with recombinant IL-17 activated the phosphorylation of mTOR, Akt, STAT3,ERK and s6k as detected by Western blotting and shown by the ratio of phosphorylated to total proteins. Note that combined use of 1,25(OH)₂D3 resulted in the most inhibitory effect on the expression of VDR, mTOR, Akt, STAT3, ERK and s6k. Bars show the mean ±SD results in 3 patients, in 1 of 3 independent experiments. **P<0.01 vs. Nill and ^‡P<0.01 vs. IL-17 1,25(OH)₂D3, 1α,25 dihydroxy-vitamin D3</p

Expression of acute and chronic injury markers in renal tubular cells treated with recombinant human IL-17.

<p>Renal tubular epithelial cell were cultured with rhIL-17 (0, 10, 50, or 100 ng/ml) for 72 hours, and the production of <b>(A)</b> IL-6 and <b>(B)</b> IL-8 was measured using ELISA. Renal tubular epithelial cell were cultured with rhIL-17 (0, 10, 50, or 100 ng/ml) for 72 hours, and the expression of <b>(C)</b><i>ACTA-2</i> and <b>(D)</b><i>CTGF</i> mRNA, relative to <i>β-actin</i>, was measured using real-time polymerase chain reaction. Bars show the means. *P<0.05 vs. Nil, ^†P<0.05 vs. IL-17 10.</p

Distribution of chemokine receptor CCR4⁺CCR6^–, CCR4^–CCR6⁺ and CCR4⁺CCR6⁺ subpopulations of CD4⁺ T lymphocytes.

<p><b>(A)</b> PBMCs were stained with anti-CD4 PE-cy7, anti-CCR4 PE, anti-CCR6 APC and anti-IL-17 FITC. CD4+ T cells were gated for further analysis. <b>(B)</b> The proportion (%) of CCR4⁺CCR6^–/CD4⁺ T cells <b>(C)</b> CCR4^–CCR6⁺/CD4⁺ T cells <b>(D)</b> CCR4⁺CCR6⁺/CD4⁺ T cells in each patient group. <b>(E)</b> After surface staining with anti-CD4, CCR4 and CCR6 mAbs, analysis of IL-17 in CD4⁺ T cell subsets by intracellular flow cytometry was done. <b>(F)</b> The proportion (%) of IL-17⁺/CCR4⁺CCR6⁺CD4⁺ T cells in each patient group. * <i>P</i><0.05 for each comparison. LTS, long term stable; CAD, chronic allograft dysfunction; ES, early stable; ESRD, end stage renal disease; HC, healthy control.</p