Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

No contribution of GSTM1 and GSTT1 null genotypes to the risk of neutropenia due to benzene exposure in Southeastern Brazil

Exposure to benzene has been associated with haematological diseases such as neutropenia (NEB) and acute myeloid leukaemia (AML). We tested whether the null genotypes of the GSTM1 and GSTT1 genes, involved in benzene inactivation, altered the risk for NEB in southeastern Brazil. Genomic DNA from 55 NEB patients and 330 controls was analysed by multiplex-polymerase chain reaction. The frequency of the GSTM1, GSTT1 and combined null genotypes was similar in patients and controls (GSTM1, 27.3% vs. 38.8%, p = 0.16; GSTT1, 25.5% vs. 19.7%, p = 0.24; GSTM1/GSTT1, 12.7% vs. 6.7%, p = 0.26; respectively). The distribution of genotype classes in NEB patients was similar to normal controls, suggesting that GSTM1 and GSTT1 null genotypes make no specific contribution to the risk of NEB. As the GSTM1 and GSTT1 null genotypes were previously associated with increased risk for AML in Brazil and elsewhere, we hypothesise that different thresholds of chemical exposure relative to distinct GSTM1 and GSTT1 genotypes may determine whether AML or NEB manifests in benzene exposed individuals from southeastern Brazil. Although indicative, our results still require support by prospective and large scale epidemiological studies, with rigorous assessment of daily chemical exposures and control of the possible contribution of other polymorphic genes involved in benzene metabolism

Further evidence of the in vivo role of erythropoietin or companion molecules induced by hypoxia on proliferation and continuing differentiation of BFU-e in PCDC

Abstract Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1–2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.</jats:p

Further evidence of the in vivo role of erythropoietin or companion molecules induced by hypoxia on proliferation and continuing differentiation of BFU-e in PCDC

Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1–2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.</jats:p