NK cell expression of receptors for specialized pro-resolving mediators

Abstract

Chronic, unresolved inflammation has been implicated in a multitude of diseases, such as cancer, asthma, and those of the immune system. Recent studies have revealed that natural killer (NK) cells may play an important role in resolution of inflammation; however, the mechanism is still unknown. NK cells were isolated and purified from the blood of healthy donors, incubated for 18 h with IL-15, and then stimulated with either a pro-inflammatory or one of two different pro-resolution cocktails, with NK cells cultured with IL-15 only serving as a control. The expression of cell surface receptors for specialized pro-resolving mediators (SPMs), i.e. Chemokine-like Receptor 1 (ChemR23), N-Formyl Peptide Receptor 2 (FPR2/ALX), and G Protein-Coupled Receptor 32 (GPR32), and the level of the SPM Annexin A1 (ANXA1) were investigated using flow cytometry and Simple Western. The concentration of Interferon Gamma (IFN-γ), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) secreted by the NK cells was measured using Enzyme-Linked Immunosorbent Assay (ELISA). Stimulation of NK cells with the cocktails did not cause a change in the expression of any of the three receptors compared to that expressed by control NK cells. However, the expression of ChemR23 and FPR2/ALX varied among NK cell subpopulations. A higher proportion of CD56dimCD16– NK cells expressed ChemR23 than that of CD56brightCD16– NK cells, but all three subpopulations of NK cells had similar mean expression of ChemR23. For FPR2/ALX, CD56dimCD16– NK cells had the highest proportion of cells expressing the FPR2/ALX receptor, and higher than both the other NK cell subpopulations. Conversely, the CD56dimCD16– NK cell subpopulation had the lowest mean expression of FPR2/ALX. Secretion of GM-CSF was inhibited by the pro-resolution cocktail when compared with both NK cells stimulated with the inflammatory cocktail and NK cells cultured with IL-15, while the pro-resolution cocktail inhibited secretion of IFN-γ only when compared with NK cells stimulated with the pro-inflammatory cocktail. There was no difference in the production of ANXA1 between NK cells treated with the different cocktails. These data show that NK cells express molecules linked to resolution of inflammation and that treating them with the pro-resolution cocktail affects their secretion of inflammatory molecules. This may be relevant in future development of therapeutics involving NK cells mediating resolution of inflammation in targeting inflammation-mediated diseases.

Krónísk bólga hefur verið bendluð við marga sjúkdóma, eins og krabbamein, astma og sjúkdóma tengda ónæmiskerfinu. Nýlegar rannsóknir sýna að náttúrulegar drápsfrumur (NK frumur) geta spilað mikilvægt hlutverk í hjöðnun bólgu en með hvaða hætti er enn óþekkt.NK frumur voru einangraðar úr blóði heilbrigðra blóðgjafa, ræktaðar í 18 klst með IL-15 og síðan örvaðar með annað hvort bólguboðefna blöndu eða einum af tveimur bólguhjöðnunarboðefna blöndum og NK frumur ræktaðar með IL-15 eingöngu notaðar sem viðmið. Tjáning yfirborðsviðtaka fyrir sérstök bólguhjöðnunar boðefni, þ.e. Chemokine-like Receptor 1 (ChemR23), N-Formyl Peptide Receptor 2 (FPR2/ALX), and G Protein-Coupled Receptor 32 (GPR32), og bólguhjöðnunarboðefnisins Annexin A1 (ANXA1) var ákvörðuð með frumuflæðisjá og Simple Western aðferðum. Styrkur Interferon Gamma (IFN-γ), og Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) seytt af NK frumum var mælt með ELISA aðferð.Örvun NK frumna með bólguboðefna eða bólguhjöðnunarboðefna blöndunum hafði ekki áhrif á tjáningu NK frumna á viðtökunum þremur. Hins vegar var tjáning á ChemR23 and FPR2/ALX breytileg milli undirhópa NK frumna. Hærra hlutfall CD56dimCD16– NK frumna tjáði ChemR23 en það sem sást hjá CD56brightCD16– NK frumum, en allar þrjár undirgerðir NK frumna höfðu svipaða meðal tjáningu á ChemR23. Hæst hlutfall NK frumna sem tjáðu FPR2/ALX var meðal CD56dimCD16– NK frumna, sem höfðu hærra hlutfall frumna jákvæðra fyrir FPR2/ALX en báðir hinir undirhópar NK frumna. Hins vegar höfðu CD56dimCD16– NK frumur lægstu meðal tjáningu á FPR2/ALX. Bólguhjöðnunarboðefna blandan hindraði seytingu NK frumna á GM-CSF borið saman við það sem NK frumur örvaðar með bólguboðefna blöndu eða ræktaðra með IL-15 seyttu. Bólguhjöðnunarboðefna blandan hindraði einnig seytingu á bólguboðefninu IFN-γ borið saman við það sem NK frumur örvaðar með bólguboðefna blöndu seyttu. Enginn munur var á myndun ANXA1 milli NK frumna örvaðra með mismunandi boðefna blöndum.Niðurstöðurnar sýna að NK frumur tjá sameindir tengdar bólguhjöðnun og að örvun með bólguhjöðnunarboðefna blöndu hindraði seytingu þeirra á bólgu sameindum. Þessar niðurstöður geta skipt máli fyrir þróun meðferðar þar sem NK frumur væru notaðar til að miðla bólguhjöðnun í bólgutengdum sjúkdómum.