Minor populations of paroxysmal nocturnal hemoglobinuria-type cells in patients with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells under the influence of pro-inflammatory mediators and oligoclonal/monoclonal T-lymphocytes. Because patients with immune-mediated BM failure display frequently paroxysmal nocturnal hemoglobinuria (PNH)-type cells in the peripheral blood (PB), we investigated the possible existence of PNH-type cells in 91 patients with CIN using flow cytometry. The patients displayed increased proportions of PNH-type glycophorin A+ /CD59dim and glycophorin A+ /CD59- red blood cells (RBCs), FLAER- /CD24- granulocytes, and FLAER- /CD14- monocytes, compared to controls (n = 55). A positive correlation was found between the proportions of PNH-type RBCs, granulocytes, and monocytes and an inverse correlation between the number of PB neutrophils and the proportions of PNH-type cell populations. The number of patients, displaying percentages of PNH-type cells above the highest percentage observed in the control group, was significantly increased among patients with skewed compared to those with normal T-cell receptor repertoire suggesting that T-cell-mediated immune processes underlie the emergence of PNH-type cells in CIN. Our findings suggest that patients with CIN display PNH-type cells in the PB at a high frequency corroborating the hypothesis that CIN belongs to the immune-mediated BM failure syndromes.

Micro-RNA analysis of renal biopsies in human lupus nephritis demonstrates up-regulated miR-422a driving reduction of kallikrein-related peptidase 4.

BACKGROUND: Aberrancies in gene expression in immune effector cells and in end-organs are implicated in lupus pathogenesis. To gain insights into the mechanisms of tissue injury, we profiled the expression of micro-RNAs in inflammatory kidney lesions of human lupus nephritis (LN). METHODS: Kidney specimens were from patients with active proliferative, membranous or mixed LN and unaffected control tissue. Micro-RNAs were quantified by TaqMan Low Density Arrays. Bioinformatics was employed to predict gene targets, gene networks and perturbed signaling pathways. Results were validated by transfection studies (luciferase assay, real-time PCR) and in murine LN. Protein expression was determined by immunoblotting and immunohistochemistry. RESULTS: Twenty-four micro-RNAs were dysregulated (9 up-regulated, 15 down-regulated) in human LN compared with control renal tissue. Their predicted gene targets participated in pathways associated with TGF-ß, kinases, NF-κB, HNF4A, Wnt/ß-catenin, STAT3 and IL-4. miR-422a showed the highest upregulation (17-fold) in active LN and correlated with fibrinoid necrosis lesions (ß = 0.63, P = 0.002). In transfection studies, miR-422a was found to directly target kallikrein-related peptidase 4 (KLK4) mRNA. Concordantly, KLK4 mRNA was significantly reduced in the kidneys of human and murine LN and correlated inversely with miR-422a levels. Immunohistochemistry confirmed reduced KLK4 protein expression in renal mesangial and tubular epithelial cells in human and murine LN. CONCLUSIONS: KLK4, a serine esterase with putative renoprotective properties, is down-regulated by miR-422a in LN kidney suggesting that, in addition to immune activation, local factors may be implicated in the disease.

Identification of novel microRNA signatures linked to human lupus disease activity and pathogenesis: miR-21 regulates aberrant T cell responses through regulation of PDCD4 expression.

OBJECTIVE: MicroRNAs (miRNAs) regulate the expression of genes involved in immune activation. A study was undertaken to characterise the miRNA signature and identify novel genes involved in the regulation of immune responses in systemic lupus erythematosus (SLE). METHODS: The expression of 365 miRNAs in peripheral blood mononuclear cells of patients with SLE and healthy controls was analysed using TaqMan Low Density Arrays. The results were validated by quantitative real-time PCR and potential target genes were identified using prediction analysis software. The effect of miR-21 on T cell function was assessed by transfection with antago-miR-21 or pre-miR-21. RESULTS: A 27-miRNA signature was identified in patients with SLE; 19 miRNAs correlated with disease activity. Eight miRNAs were deregulated specifically in T cells and four miRNAs in B cells. miR-21 was upregulated and strongly correlated with SLE disease activity (r(2)=0.92). Compared with controls, CD4 T lymphocytes from patients with SLE had higher basal and activation-induced miR-21 expression. Silencing of miR-21 reversed the activated phenotype of T cells from patients with SLE--namely, enhanced proliferation, interleukin 10 production, CD40L expression and their capacity to drive B cell maturation into Ig-secreting CD19+CD38(hi)IgD-(plasma cells. Overexpression of mMiR-21 in normal T cells led to acquisition of an activated phenotype. Investigation of putative gene- targets showed that PDCD4 (a selective protein translation inhibitor) was suppressed by miR-21 and its expression was decreased in active SLE. CONCLUSIONS: miRNAs represent potential biomarkers in SLE as their expression reflects underlying pathogenic processes and correlates with disease activity. Upregulated miR-21 affects PDCD4 expression and regulates aberrant T cell responses in human SLE.