Cellular and molecular mechanisms underlying erythropoietic response to chronic stress

Stres je sastavni deo svakodnevnog života. Psihološki stresori pokreću kompleksan odgovor organizma na stres koji utiče na različite fiziološke procese uključujući hematopoezu. Poznato je da se u bazalnim uslovima proces eritrocitopoeze odvija prevashodno u kostnoj srži, dok se u stanjima povećanih potreba organizma za eritrocitopoezom ovaj proces, nazvan stres eritrocitopoeza (SE), aktivira i u slezini. Prema najnovijim saznanjima, neadekvatna regulacija SE može rezultirati prekomernom ekspanzijom i malignom transformacijom nezrelih ćelija crvene krvne loze. Do sada je proces SE ispitivan prevashodno na mišjim modelima anemije dok su literaturni podaci o efektima hroničnog psihološkog stresa na proces eritrocitopoeze veoma oskudni. Cilj ove studije je bio: 1) da se ispita uticaj hroničnog psihološkog stresa na proces eritrocitopoeze u kostnoj srži i slezini, i 2) da se istraže ćelijski i molekularni mehanizmi regulacije ispitivanog procesa. U tu svrhu su korišćeni odrasli mužjaci miševa visokorodnih sojeva CBA i C57/BL6, kao i MIF knockout miševi koji su bili podvrgnuti restraint stresu 7 ili 14 dana uzastopno, u trajanju od 2 sata dnevno. Za detekciju progenitorskih ćelija eritrocitne loze primenjen je metod kultivacije hematopoetskih ćelija na podlozi od metilceluloze obogaćene odgovarajućim citokinima. Metodama protočne citofluorometrije i imunohistohemije izvršena je detekcija i kvantifikacija eritroblasta u ispitivanim uzorcima kostne srži i slezine. Ekspresija ciljnih proteina je analizirana primenom Western blot-a i imunohistohemije dok je relativna ekspresija ispitivanih gena određena metodom Real-Тime PCR. Naši rezultati su pokazali da hronični psihološki stres redukuje koncentraciju gvožđa i hemoglobina u krvi, i da dovodi do stimulacije eritrocitopoeze u kostnoj srži i slezini miša. Inhibicija sinteze azot monoksida u potpunosti je sprečila stimulišući efekat hroničnog stresa na progenitorske ćelije crvene krvne loze u kostnoj srži, ukazujući prvi put da ovaj signalni molekul ima značajnu ulogu u ekspanziji eritroidnih progenitora tokom hroničnog psihološkog stresa. Pored povećanog broja progenitorskih ćelija, u slezini hronično stresiranih životinja smo detektovali i znatno veći procenat eritroblasta što ukazuje da hronični psihološki stres ima izraženiji efekat na ekstramedularnu eritrocitopoezu...Stress has become an important aspect of daily life. Exposure to a psychological stressor initiates an integrated response that affects different physiological processes including hematopoiesis. The majority of steady-state erythropoiesis occurs in the bone marrow. However, recent studies have shown that the adult spleen serves as the main site of red blood cell production under different stress conditions, collectively referred to as stress erythropoiesis (SE). Furthermore, emerging evidence suggests that inappropriate activation of SE may predispose to leukemic transformation. Much of what we know about SE comes from the analysis of murine models of anemia, but the erythropoietic effects of chronic psychological stress remain largely unknown. The aim of the current study was to examine the influence of chronic psychological stress on erythropoiesis in adult bone marrow and spleen, as well as to investigate cellular and molecular mechanisms underlying the observed effects. For this purpose, adult male CBA and C57BL/6 mice were subjected to 2 hrs. daily restraint stress for 7 or 14 consecutive days. In addition, we used macrophage migration inhibitory factor (MIF)-knockout mice on a C57BL/6 background to examine whether MIF is involved in the control of stress-induced erythropoiesis. The number of erythroid progenitors was determined using colony assays, whereas CD71/Ter119 profiles of bone marrow and splenic cells were analyzed by flow cytometry. The expression of target proteins were assessed by Western blot and immunohistochemistry, while gene expression analysis was performed using real-time PCR. Our results showed that chronic exposure to restraint stress decreased the concentration of iron and hemoglobin in the blood, and resulted in markedly increased number of erythroid progenitors in murine bone marrow and spleen. Blockade of nitric oxide synthesis completely abolished the stimulatory effect of chronic restraint stress on erytrhoid progenitors in the bone marrow, suggesting a role of nitric oxide in stress-induced erythroid cell growth. Although repeated restraint stress initiated an erythroid stress response in both bone marrow and spleen, the effects on growth and maturation of erythroid cells were more prominent in the spleen, showing that chronic psychological stress stimulates mainly extramedullary erythropoiesis..