Mechanisms of interaction between mesenchymal stem cells and macrophages in the regulation of stress erythropoiesis

[S. Momčilović]

91 str.

Medicina - Molekularna medicina / Medicine - Molecular medicine

smanjene oksigenacije tkiva što za posledicu ima aktivaciju različitih mehanizama u cilju održavanja adekvatnog snabdevanja tkiva kiseonikom. Povećane potrebe organizma za eritrocitima u navedenim uslovima dovode do aktivacije stres eritrocitopoeze (SE). Tokom procesa SE dolazi do migracije eritroidnih progenitora iz koštane srži u slezinu gde pod uticajem signalnog molekula koštanog morfogenetskog proteina (engl. bone morphogenetic protein 4, BMP4) ove ćelije poprimaju karakteristike specifičnih stres eritroidnih progenitora i dolazi do njihove nagle ekspanzije. U koštanoj srži i slezini eritrocitopoeza se odvija unutar specijalizovanih eritroblastnih ostrva u kojima centralno mesto zauzimaju makrofagi koji imaju ključnu ulogu u formiranju zrelih eritrocita. U uslovima povećanih potreba za eritrocitima monociti se pod uticajem hemokina, izlučenih iz crvene pulpe slezine, iz koštane srži regrutuju u slezinu gde se diferenciraju u makrofage eritroblastne niše. Pored makrofaga, značajnu komponentu hematopoezne mikrosredine čine mezenhimalne matične ćelije (engl. mesenhimal stem cells, MSC) koje produkuju brojne citokine uključene u imunomodulaciju, proliferaciju, diferencijaciju i migraciju hematopoeznih ćelija. U zavisnosti od mikrosredine MSC i makrofagi ostvaruju interakcije posredstvom različitih signalnih molekula. Međutim, uloga makrofaga i mehanizmi njihovih interakcija sa MSC u okviru tkivno-specifične mikrosredine tokom SE do sada nisu razjašnjeni. Cilj ove studije bio je da se ispita uloga makrofaga i njihovih tkivno-specifičnih interakcija sa MSC u regulaciji procesa SE, s posebnim osvrtom na učešće azot-monoksida (engl. nitric oxide, NO), vanćelijskih nukleotida i purinergičkog signalnog sistema u ispitivanim interakcijama. U tu svrhu korišćeni su miševi, mužjaci Balb/c soja, kao i mono i ko-kulture RAW264.7 ćelija i MSC izolovanih iz koštane srži i slezine miša. Miševi su bili podvrgnuti hroničnom psihološkom stresu 7 dana uzastopno u trajanju od 2h. Deplecija makrofaga je vršena intraperitonealnom aplikacijom lipozoma koji sadrže klodronat dva dana pre početka eksperimenta radi uklanjanja rezidentnih makrofaga, a zatim svaki treći dan tokom eksperimenta, u cilju uklanjanja novostvorenih makrofaga. Ćelijske mono- i kokulture su tretirane eritropoetinom (EPO), kortikosteronom, blokatorom azot-monoksida sintetaze (engl. nitric oxide synthases, NOS) i/ili blokatorom enzimske aktivnosti molekula CD73. Deplecija makrofaga u koštanoj srži i slezini tretiranih životinja potvrđena je analizom proteina F4/80, primenom Western blota. Za detekciju progenitorskih ćelija eritrocitne loze hematopoezne ćelije su kultivisane na podlozi od metilceluloze obogaćenoj odgovarajućim citokinima. Metodama protočne citofluorometrije izvršena je detekcija i kvantifikacija eritroblasta u ispitivanim uzorcima koštane srži i slezine. Specifična aktivnost enzima određena je kolorimetrijski (detekcijom neorganskog fosfata kao proizvod enzimske reakcije). Grisova reakcija je korišćena za određivanje ukupnih nitrata i nitrita (NOx), a koncentracija vanćelijskog adenozin-trifosfata (ATP) je određena luminometrijski, primenom komercijalnog kita. Koncetracija adenozina je određena primenom tečne hromatografije visoke performanse i masene spektrometrije (engl. ultra high-performance liquid chromatography-Mass spectrometry, UHPLC-DAD MS/MS). Relativna ekspresija ispitivanih gena je određena metodom Real-Тime PCR. Rezultati su pokazali da je hronični psihološki stres smanjio broj eritrocita i nivo hemoglobina u krvi, a deplecija makrofaga je značajno pogoršala anemiju kod stresiranih životinja. Hronični psihološki stres je doveo do znatnog povećanja broja CFU-E i BFU-E ćelija u koštanoj srži i slezini, a deplecija makrofaga je suprimirala ovaj efekat stresa. Deplecija makrofaga u koštanoj srži je prevenirala efekat stresa na CD71+/Ter119+, CD71−/Ter119+ u koštanoj srži i na CD71+/Ter119+ u slezini. Takođe, u uslovima stresa, deplecija makrofaga je sprečila stresom-izazvano povećanje broja eritroidnih prekursora u okviru E1 i E2 subpopulacije u koštanoj srži, kao i povećanje broja E1, E2 i E3 ćelija u slezini...

In conditions of disturbed homeostasis, such as bleeding, stress, inflammation, etc., tissue hypoxia occurs, leading to the activation of various mechanisms to maintain adequate oxygen supply. Increased demand for erythrocytes in these conditions results in the activation of stress erythropoiesis (SE). During the SE process, erythroid progenitors migrate from the bone marrow to the spleen, where these cells affected with bone morphogenetic protein 4 (BMP4) gain characteristics specific to stress erythroid progenitors and extend rapidly. In the bone marrow and spleen, erythropoiesis takes place within specialized erythroblastic islands, with macrophages playing a crucial role in the formation of mature erythrocytes. In conditions of increased erythrocyte demand, monocytes are recruited to the spleen under the influence of chemokines released from the red pulp, where they differentiate into macrophages within the erythroblastic niche. Besides macrophages, a significant component of the hematopoietic microenvironment is composed of mesenchymal stem cells (MSC), which produce numerous cytokines involved in immunomodulation, proliferation, differentiation, and migration of hematopoietic cells. Depending on the microenvironment, MSCs and macrophages establish interactions through various signaling molecules. However, the role of macrophages and the mechanisms of their interactions with MSCs within the tissue-specific microenvironment during SE have not been clarified yet. The aim of this study was to investigate the role of macrophages and their tissue-specific interactions with MSCs in the regulation of stress erythropoiesis, with a particular focus on the involvement of nitric oxide (NO), extracellular nucleotides, and the purinergic signaling system in the examined interactions. For this purpose, male Balb/c mice were used, as well as mono- and co-cultures of RAW264.7 cells and MSC isolated from the bone marrow and spleen of mice. The mice were subjected to chronic psychological stress for 7 consecutive days, 2 hours per day. Macrophage depletion was performed by intraperitoneal administration of liposomes containing clodronate two days before the start of the experiment to remove resident macrophages, followed by administration every third day of the experiment to eliminate newly formed macrophages. Cell mono- and co-cultures were treated with erythropoietin (EPO), corticosterone, a nitric oxide synthase (NOS) inhibitor, and/or a CD73 inhibitor. Macrophage depletion in the bone marrow and spleen of treated animals was confirmed by analyzing the F4/80 protein using Western blotting. To detect erythroid progenitor cells, hematopoietic cells from the erythroid lineage were cultured on methylcellulose-enriched substrates with appropriate cytokines. Flow cytometry methods were used for the detection and quantification of erythroblasts in the examined samples of bone marrow and spleen. Enzyme-specific activity was determined colorimetrically by detecting inorganic phosphate as the product of the enzymatic reaction. Gris reaction was used to determine total nitrates and nitrites (NOx), while the concentration of extracellular adenosine triphosphate (ATP) was determined luminometrically using a commercial kit. Adenosine concentration was determined using high-performance liquid chromatography and Mass spectrometry (UHPLC-DAD MS/MS). Relative expression of the examined genes was determined by Real-Time PCR. The results showed that chronic psychological stress reduced the number of red blood cells and hemoglobin levels in the blood, and macrophage depletion significantly alered anemia in stressed animals. Chronic psychological stress led to a significant increase in the number of colony-forming units-erythroid (CFU-E) and burst-forming units-erythroid (BFU-E) cells in the bone marrow and spleen, and macrophage depletion suppressed this stress effect. Macrophage depletion in the bone marrow prevented the stress effect on CD71+/Ter119+ and CD71+/Ter119+ populations in the bone marrow and spleen, respectively. Also, under stress conditions, macrophage depletion prevented stress-induced increases in the number of immature erythroid precursors within the E1 and E2 subpopulations in the bone marrow, as well as the increased number of E1, E2, and E3 cells in the spleen...

stres eritrocitopoeza, makrofagi, mezenhimalne stromalne ćelije, azot monoksid, purinergički signalni sistem

stress erythropoiesis, macrophages, mesenchymal stem cells, nitric oxide, purinergic signaling system

612.119:577.2(043.3)

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