Autophagy mediated activation of Relb/p52 responsible for the reprogramming of tumour associated macrophages

Abstract

INTRODUCTION: The heterogeneity of macrophages has underlined their diverse roles in disease progression upon exposure to different environmental cues. High population of alternatively activated M2 macrophages surrounding tumor microenvironment has been implicated to be associated with tumor growth, expansion, invasion and migration. Re-skewing of M2 macrophages towards tumoricidal M1-like phenotype defines a microenvironment that favors tumor destruction. In this study, we demonstrate autophagy mediated RelB/p52 activation responsible in reprogramming of phenotypic states of tumor associated macrophages further leading to regression of hepatocellular carcinoma growth. MATERIAL AND METHODS: Bone marrow isolated mononuclear cells were differentiated by either culturing with M-CSF or tumor supernatant (TSN) for 7 days. Expressions of mRNAs was detected by quantitative real time PCR; phenotypic change of macrophages was detected by flow cytometry analysis; protein expression was detected by immunoblotting assay; gene silencing was performed by RNA interference. RESULTS: We observed increased of LC3-II/LC3-I ratio and LC3 punctuation in IFNg-induced M1-like macrophages, while not in IL-4 induced M2-like and TSN cultured macrophages. Blockade of autophagy through silencing of Atg5 and intervention of pharmacological inhibitor, balfilomycin A1 ameliorated the autophagy mediated reprogramming of tumor associated macrophages towards M1 phenotype. It was also accompanied with reduced of proinflammatory and increased of anti-inflammatory genes. Simultaneously, increased expression of RelB and p52 is observed in M1-like macrophages while detected low in M2 and TSN cultured macrophages. Atg5 depletion further blocked the RelB/p52 nuclear translocation and its trans-activated CCL9 and CXCL12 genes in M1-like macrophages. Depletion of RelB induced the reprogramming of M1-like macrophages towards M2 phenotype and increased anti-inflammatory M2-mediated cytokines. Furthermore, we observed downregulation of TRAF2, the negative regulator of RelB/p52 pathway in M1-like macrophages and depletion of Atg5 restored the expression of TRAF2. The co-culturing of Atg5 silencing macrophage supernatant with HCC cells blocked the migration and proliferation of HCC cells. CONCLUSION: Altogether, our findings unveil the role of autophagy in mediating RelB/p52 pathway regulates macrophages reprogramming which associated with HCC tumor suppression.