From the above results we concluded that increased expression of CIITA, KDM6B, and NCOA1 and decreased expression of KAT2A, PRMT7, SETD6, and SMYD3 was independent of any effects on proliferation. To begin to understand the impact of these changes on macrophage phenotype, we chose to further investigate KDM6B, which was the only transcriptional enhancer that was increased at the mRNA level by both IFN-γ and IL-4. Given that the action of KDM6B is demethylation of lysine 27 on histone3, which is associated with increased gene transcription (13), this upregulation might be expected to promote transcriptional responses to both cytokines. Conversely, inhibition and silencing of KDM6B might decrease gene expression associated with IFN-γ and IL-4. To narrow down the search for those genes regulated by KDM6B, either on its own or in combination with KDM6A, we first performed a microarray study of transcripts upregulated by IFN-γ or IL-4 in the presence and absence of the combined KDM6A and B inhibitor GSK-J4 (39). From preliminary time course and dose-response studies (results not shown), 6 h exposure was sufficient and 60 µM GSK-J4 was chosen because it significantly suppressed TNFα induction by lipopolysaccharide (LPS) by approximately 70% [confirming previous results (39)]. CD206 induction by IL-4 was also inhibited, albeit by only 30% (both p < 0.05), whereas a housekeeping gene, 36B4, was not affected. From these pre-validated samples, the transcriptomic analysis showed that IFN-γ significantly upregulated 906 and IL-4 upregulated 271 transcripts after 6 h (a complete gene list is deposited under GSE83957). Only 62 of these (6%) were upregulated by both IFN-γ and IL-4, which confirms the differential phenotypes stimulated by these two cytokines, as demonstrated more extensively previously (1). This data also emphasizes how unusual KDM6B is in being upregulated by both IFN-γ and IL-4. Of the 831 IFN-γ upregulated transcripts recognized by the genevenn program used to generate Venn diagrams, 181 (22%) were significantly decreased by the additional presence of GSK-J4 (Figure 8A). Using GOE and KEGG pathway analysis, these genes were associated with several aspects of immune cell function and transcriptional activation (Tables 3 and 4). Of the 254 IL-4 upregulated transcripts only 28 (11%) were significantly reduced by the additional presence of GSK-J4 (Figure 8B). There were insufficient genes in this cluster to perform GOE or KEGG pathway analysis. Only two transcripts (C17orf87 and LOC650919), neither associated with a known function, were common to both lists. The genes most inhibited by GSK-J4 in the presence of either IFN-γ or IL-4 are illustrated by heat maps in Figures 8C,D, respectively, with the details of these and further genes listed in Table 5. Heading the list of IFN-γ upregulated, GSK-J4 downregulated genes were CCL7 and CCL8, which are known genes associated with activation by IFN-γ (40). Heading the list of IL-4 upregulated, GSK-J4 downregulated gene was CD209, which is an established IL-4 responsive gene (41). A selection of the more abundant transcripts that were inhibited at least twofold by GSK-J4, were chosen for further analysis. Because there were so few abundant IL-4 stimulated, GSK-J4 inhibited transcripts in the array, we also included CD206, which was used for initial sample validation but just failed to reach significance in the array experiment (i.e., a false negative). First, upregulation by IFN-γ or IL-4 and its reversal by GSK-J4 was confirmed by RT-qPCR (Figures 8E,F, respectively). GSK-J4 is non-selective for KDM6A and KDM6B (39). Hence, to distinguish the roles of KDM6A and KDM6B, the effects of silencing one, the other or both was investigated by using shRNA. Given that silencing is difficult in primary macrophages, we used adenovirus-mediated delivery of shRNAs selective for KDM6A or KDM6B and compared these to delivery of a control adenovirus that expressed shRNA against firefly luciferase (shLUC). The housekeeping gene, 36B4, was also studied as a further control. Based on mRNA levels, silencing of KDM6A and B was highly selective, albeit incomplete at the maximum tolerable adenovirus dose (Figure 9A). Specificity and efficacy was confirmed at the protein level for KDM6A (Figure 9B) but no suitable antibody is available for KDM6B. The effects of KDM6A and B silencing were measured in the presence of IFN-γ or IL-4. From these results, the induction of CCL7 by IFN-γ depended selectively on KDM6A, whereas that of VAMP5 required KDM6A and B redundantly (Figure 9C). The induction of CD206 by IL-4 also required KDM6A and B redundantly, whereas that of PALLD depended selectively on KDM6B (Figure 9C). We concluded that a subgroup of transcripts upregulated by IFN-γ or IL-4 depended on KDM6B, either on its own or redundantly with KDM6A.