CD8⁺ Treg cells associated with decreasing disease activity after intravenous methylprednisolone pulse therapy in lupus nephritis with heavy proteinuria.

We focus on the role of CD8(+) Treg cell in Intravenous methyl-prednisolone (IVMP) pulse therapy in forty patients with active Class III/IV childhood lupus nephritis (LN) with heavy proteinuria. IVMP therapy for five days. From peripheral blood mononuclear cells (PBMCs) and renal tissues, we saw IVMP therapy definitely restoring both CD4(+)CD25(+)FoxP3(+) and CD8(+)CD25(+)Foxp3(+) Treg cell number plus greater expression with intracellular IL-10 and granzyme B in CD8(+)FoxP3(+) Treg from PBMCs. IVMP-treated CD8(+)CD25(+) Treg cells directly suppressed CD4(+) T proliferation and induced CD4(+)CD45RO(+) apoptosis. Histologically, CD4(+)FoxP3(+) as well as CD8(+)FoxP3(+) Treg cells appeared in renal tissue of LN patients before IVMP by double immunohistochemical stain. CD8(+)FoxP3(+) Treg cells increased in 10 follow-up renal biopsy specimens after IVMP. Reverse correlation of serum anti-C1q antibody and FoxP3(+) Treg cells in PBMNCs (r = -0.714, P<0.01). After IVMP, serum anti-C1q antibody decrease accompanied increase of CD4(+)FoxP3(+) Treg cells. CD8(+)Treg cells reduced interferon-r response in PBMCs to major peptide autoepitopes from nucleosomes after IVMP therapy; siRNA of FoxP3 suppressed granzyme B expression while decreasing CD8(+)CD25(+)Treg-induced CD4(+)CD45RO(+) apoptosis. Renal activity of LN by SLEDAI-2k in childhood LN was significantly higher than two weeks after IVMP (P<0.01). CD8(+)FoxP3(+) Treg cells return in post-IVMP therapy and exert crucial immune modulatory effect to control autoimmune response in LN.DMR97-IRB-259

The attenuation of renal fibrosis by histone deacetylase inhibitors is associated with the plasticity of FOXP3+IL-17+ T cells

Abstract Background The histone deacetylase (HDAC) inhibitor, which has potential effects on epigenetic modifications, had been reported to attenuate renal fibrosis. CD4+ forkhead box P3 (FOXP3)+ T regulatory (Treg) cells may be converted to inflammation-associated T helper 17 cells (Th17) with tissue fibrosis properties. The association between FOXP3+IL-17+ T cells and the attenuation of renal fibrosis by the HDAC inhibitor is not clear. Methods This study evaluated the roles of the HDAC inhibitor, Treg cells and their differentiation into Th17 cells, which aggravate chronic inflammation and renal fibrosis in a unilateral ureteral obstruction (UUO) mouse model. The study groups included control and UUO mice that were monitored for 7, 14 or 21 days. Results Juxtaglomerular (JG) hyperplasia, angiotensin II type 1 receptor (AT1R) expression and lymphocyte infiltration were observed in renal tissues after UUO but were decreased after trichostatin A (TSA) treatment, a HDAC inhibitor. The number of CD4+FOXP3+ T cells increased progressively, along with the number of FOXP3+interleukin (IL)-17+ T cells, after 14 days, and their numbers then progressively decreased with increasing CD4+IL-17+ T cell numbers, as demonstrated by double immunohistochemistry. Progressive renal fibrosis was associated with the loss of CD4+FOXP3+IL-17+ T cells in splenic single-cell suspensions. FOXP3+IL-17+ T cells expressed TGF-β1 both in vitro and in vivo, and TGF-β1 expression was significantly knockdown by IL-17 siRNA in vitro. These cells were found to play a role in converting Tregs into IL-17- and TGF-β1-producing cells. Conclusions TSA treatment decreased JG hyperplasia, the percentage of FOXP3+IL-17+ cells and the degree of fibrosis, suggesting that therapeutic benefits may result from epigenetic modifications

Montelukast Increased IL-25, IL-33, and TSLP via Epigenetic Regulation in Airway Epithelial Cells

The epithelium-derived cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) are important mediators that initiate innate type 2 immune responses in asthma. Leukotriene receptor antagonists (LTRAs) are commonly used to prevent asthma exacerbations. However, the effects of LTRAs on epithelium-derived cytokines expression in airway epithelial cells are unclear. This study aimed to investigate the effects of LTRAs on the expression of epithelium-derived cytokines in human airway epithelial cells and to explore possible underlying intracellular processes, including epigenetic regulation. A549 or HBE cells in air-liquid interface conditions were pretreated with different concentrations of LTRAs. The expression of epithelium-derived cytokines and intracellular signaling were investigated by real-time PCR, enzyme-linked immunosorbent assay, and Western blot. In addition, epigenetic regulation was investigated using chromatin immunoprecipitation analysis. The expression of IL-25, IL-33, and TSLP was increased under LTRAs treatment and suppressed by inhaled corticosteroid cotreatment. Montelukast-induced IL-25, IL-33, and TSLP expression were mediated by the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways and regulated by histone H3 acetylation and H3K36 and H3K79 trimethylation. LTRAs alone might increase inflammation and exacerbate asthma by inducing the production of IL-25, IL-33, and TSLP; therefore, LTRA monotherapy may not be an appropriate therapeutic option for asthma

Baseline characteristics.

<p>Mean±SD reported for quantitative variables, F: female, M: Male. Cr:creatinine, angiotensin-converting enzyme inhibitors (ACEIs), angiotensin-receptor blocker (ARBs).</p

Intracellular IL-10 and granzyme B levels in CD8⁺CD25⁺ Treg cells before and after IVMP.

<p>PBMCs were stimulated with anti-CD3 mAb for five days, followed by stimulation of PMA (10 ng/ml) plus ionomycin (1 µg/ml) for the last five hours, with addition of brefeldin A (10 µg/ml) for the final hour. Intracellular expression of IL-10 and granzyme B was measured by gating in CD8⁺CD25⁺ T cells, using flow cytometry. Isotype control (dotted line). (a) Results of 30 paired experiments for IL-10 (b) and granzyme B (c) production by PBMCs (* <i>p</i><0.05).</p

IL-25 Induced ROS-Mediated M2 Macrophage Polarization via AMPK-Associated Mitophagy

Interleukin (IL)-25 is a cytokine released by airway epithelial cells responding to pathogens. Excessive production of reactive oxygen species (ROS) leads to airway inflammation and remodeling in asthma. Mitochondria are the major source of ROS. After stress, defective mitochondria often undergo selective degradation, known as mitophagy. In this study, we examined the effects of IL-25 on ROS production and mitophagy and investigated the underlying mechanisms. The human monocyte cell line was pretreated with IL-25 at different time points. ROS production was measured by flow cytometry. The involvement of mitochondrial activity in the effects of IL-25 on ROS production and subsequent mitophagy was evaluated by enzyme-linked immunosorbent assay, Western blotting, and confocal microscopy. IL-25 stimulation alone induced ROS production and was suppressed by N-acetylcysteine, vitamin C, antimycin A, and MitoTEMPO. The activity of mitochondrial complex I and complex II/III and the levels of p-AMPK and the mitophagy-related proteins were increased by IL-25 stimulation. The CCL-22 secretion was increased by IL-25 stimulation and suppressed by mitophagy inhibitor treatment and PINK1 knockdown. The Th2-like cytokine IL-25 can induce ROS production, increase mitochondrial respiratory chain complex activity, subsequently activate AMPK, and induce mitophagy to stimulate M2 macrophage polarization in monocytes

Disease activity and laboratory variables in both groups before and two weeks after IVMP treatment.

*<p>P<0.01,</p>**<p>P<0.05 compared to pre-IVMP pulse therapy.</p

Number of interstitial CD3⁺, FoxP3⁺, CD4FoxP3⁺ and CD8⁺FoxP3⁺ cells in renal biopsy.

*<p>P<0.05 vs CD3+ cell group, data shown as mean±SEM.</p

CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD25⁺FoxP3⁺ Treg cells before and after IVMP pulse therapy.

<p>(a) PBMCs from LN patients during IVMP pulse therapy were stimulated with anti-CD3 mAb stimulation (5 ug/ml) and IL-2 (10 U/ml) for five days, and cells with intracellular expression of FoxP3 were analyzed for CD4⁺CD25⁺ and CD8⁺CD25⁺ T cells, representative figures shown. Analysis of CD4⁺CD25⁺FoxP3 Treg cells (b) and CD8⁺CD25⁺FoxP3 Treg cells (c) in PBMCs before and after IVMP pulse therapy by flow cytometry. Bars represent mean ± SD.</p