Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer (2021)
Figure 4

Figure 4. IRE1α-TRAF2-NF-κB axis is essential for polarizing M2 to M1-like macrophage

A, B Deficiency of Ern1 reduces the expression of inflammatory related genes. Wild-type and Ern1-/- Raw264.7 cells (A) or BMDMs (B) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). The mRNA of Il-1β and Il-6 were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).

C. Inhibition of IRE1α kinase activity represses luciferase of Carfilzomib treated M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs derived from Il-1β-luciferase transgenic mice were differentiated into M2 macrophages by IL-4 (20 ng/mL). M2 macrophages were treated by different inhibitors of IRE1α including Kira6 (inhibitor of IRE1α kinase activity, 100 nM), 4μ8c (inhibitor of IRE1α Rnase activity, 100 nM) for 1 hour, followed by stimulation with Carfilzomib (1 μM) for 12 hours. Luciferase activity was then monitored. The data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).

D. Kira6 inhibits Carfilzomib induced Il-1β-luciferase in M2 macrophages in a dose-dependent manner. Il-1β-luciferase BMDMs was differentiated into M2 macrophages by IL-4 (20 ng/mL), treated by Kira6 at indicated concentrations (0， 50 nM, 100 nM, 200 nM, 400 nM, 800 nM, 1600 nM) for 1 hour, and then stimulated by Carfilzomib (1 μM) for 12 hours. Luciferase activity was measured. The data are means ± SD of three independent experiments.

E. Kira6 inhibits the expression of ER stress related genes and inflammatory related genes. BMDMs was treated by IL-4 (20 ng/mL) for 24 hours, treated with Kira6 (100 nM) for 1 hour, and then stimulated by Carfilzomib (1 μM) for 6 hours. RNA was extracted from cells and the expression of Bip, Chop, Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).

F, G. Carfilzomib has no effect on splicing of XBP1 in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated with DMSO, Carfilzomib (1 μM) or TUN (500 nM). The mRNA expression of sXBP1 and unXBP1 were detected through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).

H. Carfilzomib increases the expression of total IRE1α as well as promotes the phosphorylation of IRE1α. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM) for indicated time. Whole Cell lysates (WCL) were analyzed by immunoblots (IB) with the indicated antibodies.

I. Carfilzomib promotes the oligomerization of IRE1α in macrophages. Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM) for 2 hours. The cells were fixed and stained with anti-IRE1α antibody and fluorochrome-conjugated secondary antibody, and then subjected to confocal microscopy. Representative images show the aggregation of IRE1α (green). Scale bar: 10 μM.

J. Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages. Raw264.7 cells were pretreated by IL-4 (20ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for 2 or 4 hours. Coimmunoprecipitation (Co-IP) and IB were performed with indicated antibodies.

K. Carfilzomib activates NF-κB promoter in Raw264.7 cells. Raw264.7 cells were pretreated with or without IL-4 (20 ng/mL) for 24 hours, NF-κB reporter and TK were co-transfected for overnight, then stimulated by DMSO or Carfilzomib (1 μM) for 12 hours before luciferase assay performed. The data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).

L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for indicated time points. WCL were analyzed by IB with indicated antibodies.