RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma.

Radiotherapy induces DNA damage, resulting in cell-cycle arrest and activation of cell-intrinsic death pathways. However, the radioresistance of some tumour entities such as malignant melanoma limits its clinical application. The innate immune sensing receptor retinoic acid-inducible gene I (RIG-I) is ubiquitously expressed and upon activation triggers an immunogenic form of cell death in a variety of tumour cell types including melanoma. To date, the potential of RIG-I ligands to overcome radioresistance of tumour cells has not been investigated. Here, we demonstrate that RIG-I activation enhanced the extent and immunogenicity of irradiation-induced tumour cell death in human and murine melanoma cells in vitro and improved survival in the murine B16 melanoma model in vivo. Transcriptome analysis pointed to a central role for p53, which was confirmed using p53-/- B16 cells. In vivo, the additional effect of RIG-I in combination with irradiation on tumour growth was absent in mice carrying p53-/- B16 tumours, while the antitumoural response to RIG-I stimulation alone was maintained. Our results identify p53 as a pivotal checkpoint that is triggered by RIG-I resulting in enhanced irradiation-induced tumour cell death. Thus, the combined administration of RIG-I ligands and radiotherapy is a promising approach to treating radioresistant tumours with a functional p53 pathway, such as melanoma.

Tenascin-C restricts reactive astrogliosis in the ischemic brain.

Cellular responses in glia play a key role in regulating brain remodeling post-stroke. However, excessive glial reactivity impedes post-ischemic neuroplasticity and hampers neurological recovery. While damage-associated molecular patterns and activated microglia were shown to induce astrogliosis, the molecules that restrain astrogliosis are largely unknown. We explored the role of tenascin-C (TnC), an extracellular matrix component involved in wound healing and remodeling of injured tissues, in mice exposed to ischemic stroke induced by transient intraluminal middle cerebral artery occlusion. In the healthy adult brain, TnC expression is restricted to neurogenic stem cell niches. We previously reported that TnC is upregulated in ischemic brain lesions. We herein show that the de novo expression of TnC post-stroke is closely associated with reactive astrocytes, and that astrocyte reactivity at 14 days post-ischemia is increased in TnC-deficient mice (TnC-/-). By analyzing the three-dimensional morphology of astrocytes in previously ischemic brain tissue, we revealed that TnC-/- reduces astrocytic territorial volume, branching point number, and branch length, which are presumably hallmarks of the homeostatic regulatory astrocyte state, in the post-acute stroke phase after 42 days. Interestingly, TnC-/- moderately increased aggrecan, a neuroplasticity-inhibiting proteoglycan, in the ischemic brain tissue at 42 days post-ischemia. In vitro in astrocyte-microglia cocultures, we showed that TnC-/- reduces the microglial migration speed on astrocytes and elevates intercellular adhesion molecule 1 (ICAM1) expression. Post-stroke, TnC-/- did not alter the ischemic lesion size or neurological recovery, however microglia-associated ICAM1 was upregulated in TnC-/- mice during the first week post stroke. Our data suggest that TnC plays a central role in restraining post-ischemic astrogliosis and regulating astrocyte-microglial interactions.

Tenascin-C preserves microglia surveillance and restricts leukocyte and, more specifically, T cell infiltration of the ischemic brain.

As an endogenous activator of toll-like receptor-4 (Tlr4), the extracellular matrix glycoprotein tenascin-C (TnC) regulates chemotaxis, phagocytosis and proinflammatory cytokine production in microglia. The role of TnC for ischemic brain injury, post-ischemic immune responses and stroke recovery has still not been evaluated. By comparing wild type and TnC-/- mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO), we examined the effects of TnC deficiency for ischemic injury, neurological deficits, microglia/macrophage activation and brain leukocyte infiltration using behavioural tests, histochemical studies, Western blot, polymerase chain reaction and flow cytometry. Histochemical studies revealed that TnC was de novo expressed in the ischemic striatum, which contained the infarct core, and overlapped with the area of strongest accumulation of Iba1 + microglia/macrophages. TnC deficiency increased overall Iba1 immunoreactivity in the perilesional cortex, suggesting that TnC might restrict the distribution of microglial cells to the infarct core. By analysing microglial morphology in 3D we found that the post-ischemic loss of microglial cell territory, branching and volume at 3 and 7 days post-ischemia was amplified in the brains of TnC deficient compared with wild type mice. Microglial cell number was not different between genotypes. Hence, TnC deficiency reduced tissue surveillance by microglial cells. Concomitantly, the number of infiltrating leukocytes and, more specifically, T cells was increased in the ischemic brain parenchyma of TnC deficient compared with wild type mice. Ischemic injury and neurological deficits were not affected by TnC deficiency. We propose that the reduced microglia surveillance in TnC deficient mice might favour leukocyte accumulation in the ischemic brain.