Human IFN-?3 and IFN-?4 show distinct molecular phenotypes in macrophages

CD14+ cells were used to obtain M1 or M2-MDMs in presence of recombinant human IFN-?3 or IFN-?4. Briefly, four Caucasian donors (n=4; one female and three males) in between 31-41 y of age, were used to obtain highly homogenous CD14+ cells; these were purchased from a commercial source. The CD14+ cells were incubated with GM-CSF (for M1-MDM) or M-CSF (for M2-MDM) in presence or absence of IFN-?3 (at 50 ng/ml) or IFN-?4 (at 6 µg/ml) for six days, and then the cells were fully washed off any residual growth factors or IFNs. After six days of differentiation, the macrophages were matured with LPS for 24 h, then the total cellular RNA was isolated and RNA-seq was performed at QuickBiology, Psadena, CA, USA Summary of the study: Type III interferons or IFN-?s have emerging roles beyond antiviral defense at barrier surfaces. IFN-?3 and IFN-?4 are genetically associated with several infectious and inflammatory diseases; however, strong linkage at the IFNL locus circumvents a clearer understanding of their differential influence on the disease phenotypes. Comparative studies to examine the effect of IFN-?3 and IFN-?4 on immune cells are lacking. In this study, we used human CD14+ monocytes differentiated into macrophages (MDMs) in presence of IFN-?3 or IFN-?4 under M1 or M2 conditions, and after maturation with lipopolysaccharides, to perform RNA-seq analysis in order to understand the changes in molecular phenotypes that may have been induced by the two IFN-?s. Firstly, we found that IFN-?4 can induce several genes, both unique and common to those induced by IFN-?3, especially in M1-MDMs; our analysis showed that close to 870 genes (in both cell types) were reciprocally regulated by IFN-?3 and IFN-?4. Secondly, the induction of differentially expressed gene pattern suggested that IFN-?3 has a stronger effect on M2-MDMs compared to M1-MDMs while the reverse is true for IFN-?4. Overall design: Sequecing RNA from MDMs Grant ID: IA/I/17/1/503122 Grant title: Functional interrogation of interferon lambda 4 (IFN-L4) in human health and disease Funding agency: DBT/Wellcome Trust India Alliance Fellowship