Figure 1.
Macrophages differentiated in the presence of IDR-1018 showed an intermediate cytokine response profile when compared to M1 and M2 macrophages.
Adherent monocytic cells were differentiated into macrophages in the presence of IFNγ (M1), M-CSF (M2), IDR-1018 alone or in combination with M-CSF (1018+M2), or left untreated (M0). Macrophages were then left unstimulated or stimulated with LPS for 4 or 24 hours after which, the cytokine responses were measured by ELISA (A) and RT-qPCR (B). The data was analyzed for significant differences between the treatments and the M1 phenotype. Mean ± SD results are presented and are representative of 4 biological replicates. ***, P<.0.0001; **, P<0.01; *, P<0.05.
Figure 2.
Macrophages differentiated in the presence of IDR-1018 exhibited a chemokine profile different to that of M2 macrophages.
Adherent monocytic cells were differentiated into macrophages in the presence of IFNγ (M1), M-CSF (M2), IDR-1018 alone or in combination with M-CSF (1018+M2), or left untreated (M0). These macrophages were then stimulated ± LPS. Twenty four hours post stimulation; the chemokine responses were measured by ELISA and analyzed for significant differences when compared to the M1 phenotype. Raw values were normalized to M0 Macrophages. Mean ± SD results are presented and are representative of 4 biological replicates. **, P<0.01; *, P<0.05.
Figure 3.
Differentiation of macrophages in the presence of IDR-1018 induced the expression of wound healing associated genes.
Adherent monocytic cells were differentiated into macrophages in the presence of IFNγ (M1), M-CSF (M2), IDR-1018 alone or in combination with M-CSF (1018+M2), or left untreated (M0). These macrophages were then stimulated ± LPS. Four hours post-stimulation, transcriptional changes in wound healing associated genes were measured by RT-qPCR, and analyzed for significant differences when compared to the M1 phenotype. Mean ± SD results are presented and are representative of 4 biological replicates. **, P<0.01; *, P<0.05.
Figure 4.
IDR-1018 differentiated macrophages maintained plasticity as they could return to a pro-inflammatory state.
Macrophages were differentiated as described previously and treated ± LPS. In addition, macrophages differentiated in the presence of IDR-1018 alone or in combination with M-CSF, were subsequently treated with M1-inducing IFNγ for 24 hours then challenged with LPS. Four hours post-challenge, cytokine responses were analyzed by ELISA. Mean ± SD results are presented and are representative of 3 biological replicates. *, P<0.05.
Figure 5.
IDR-1018 treated monocytes and IDR-1018 differentiated macrophages expressed transcription factors important for the development of alternative (M2) macrophages.
(A) Macrophages were differentiated in the presence of IDR-1018 or the combination of IDR-1018 and M2-inducing factor M-CSF, and RT-qPCR was performed to analyze the expression of different M2 specific transcription factors. (B) Additionally, monocytes were stimulated with IDR-1018 (5 ug/ml) for 4 hours. In both cases, RNA was isolated and the expression of transcription factors was analyzed by RT-qPCR. Mean ± SD results are presented and are representative of 3 biological replicates. *, P<0.05.