Cell death and differentiation. macrophages with TGF did not affect expression of iNOS or arginase, nor was it able to change the ability of IFNg and LPS to induce iNOS or IL-4 to induce arginase [42]. Thus, like Gas6, treatment of macrophages with TGF1 resulted in altered macrophage cytokine responses without changing expression Swertiamarin of the prototypical effector molecules of M1 or M2 differentiated cells. The presence of a specific TGFR inhibitor was able to inhibit Swertiamarin the conversion to IL-10 production by irradiated cancer cells (Figure ?(Figure4c);4c); however, the TGFR inhibitor was not able to restore TNF production by macrophages (Figure ?(Figure4c).4c). To test the combination with Mertk inhibition, we co-cultured irradiated cancer cells with macrophages in the presence of a TGFR inhibitor, a Mertk-Fc blocking antibody or the combination. We demonstrated that irradiated cancer cells redirect macrophages to secrete suppressive cytokines, and both Mertk-Fc and TGFR inhibitor partially block suppressive cytokine secretion (Figure ?(Figure4d),4d), but that the combination of the TGFR inhibitor together with a blocking MertkFc fusion protein was able to completely inhibit the co-culture induced switch to IL-10 production and importantly was able to restore TNF production in response to LPS stimulation (Figure ?(Figure4d).4d). These data demonstrate that Mertk ligation and TGF each individually prevent proinflammatory differentiation of Rabbit Polyclonal to Galectin 3 macrophages, and combined blockade permits proinflammatory differentiation even in the presence of dying cancer cells. Open in a separate window Figure 4 The combination of Mertk knockout and TGF inhibition restores proinflammatory function of macrophages in the presence of irradiated cancer cellsa. C57BL/6 wild-type or C57BL/6 Mertk?/? mice were challenged with Panc02 pancreatic adenocarcinoma and tumors were left untreated (we treated wild type or Mertk knockout mice with the orally bioavailable small molecule TGFR1 inhibitor SM16 [42] for two weeks following treatment with radiation therapy (Figure ?(Figure5).5). As before, tumor growth and therapy were identical in wild-type and Mertk?/? mice (Figure ?(Figure5)5) and as we have previously shown, TGFR inhibition alone did not significantly alter tumor growth [42]. When combined with radiation therapy, TGFR inhibition extended survival in wild-type mice but in Mertk?/? mice TGFR inhibition was dramatically more effective and resulted in tumor cures (Figure ?(Figure5b).5b). Importantly, this combination of Mertk?/? and TGFR inhibition did not affect tumor growth unless radiation therapy was present, suggesting that the large-scale cell death induced by radiation therapy was required to initiate this response. During tumor rejection, Mertk?/? mice treated with TGFR inhibitors frequently exhibited either moist or dry desquamation in the radiation field that was not seen to any significant degree in Swertiamarin any other group. This increased toxicity of radiation therapy resolved over time and resulted in a scarred treatment site but no other detectable problems in survivor mice. These data demonstrate that radiation therapy in the presence of combined loss of Mertk and TGFR signaling is curative even in a highly unresponsive pancreatic adenocarcinoma, and demonstrates that therapeutically manipulating the macrophage response to dying cells in the tumor environment is a potential strategy to enhance the efficacy of radiation therapy. Open in a separate window Figure 5 The Swertiamarin combination of Mertk knockout and TGF inhibition permits tumor cure following RT of poorly immunogenic tumorsa. C57BL/6 wild-type or b. C57BL/6 Mertk?/? mice were challenged with Panc02 pancreatic adenocarcinoma and Swertiamarin tumors were left untreated or treated on d14 with 20Gy x3 of focal radiation to the tumor (dashed lines). Mice were additionally treated with control food or food containing the orally bioavailable TGF inhibitor SM16 (shading). Graphs show tumor size in individual mice: i) untreated; ii) RT alone; iii) SM16 alone; iv) RT+SM16; v) Overall survival. Results are representative of two or more experimental repeats of.