Transcriptional and translational regulation underlying LPS/IFN-γ-mediated macrophage polarization

Abstract

As the central component of innate immune system, macrophages’ functions include killing of invading pathogens through cytotoxic inflammatory response, phagocytosis and regulation of the immune system activities. They reflect cellular heterogeneity and are classified as M1 (classical activation) upon activation by LPS, IFN-γ or TNFα and M2 (alternative activation) when activated by IL-4/13. Several genes expressions occur during macrophage polarization that are controlled by multiple transcriptional and translational mechanisms leading to their distinct phenotypic forms and systemic innate immune responses. The present study underscores the important contributions of IFN-γ and/ or LPS in murine macrophage polarization. I stimulated BMDMs with LPS or LPS + IFN-γ and confirmed the polarized macrophage populations using flow cytometry. During LPS or LPS + IFN-γ response at early or late time point, canonical M1 genes (Nos2 and TNFα) expression were validated with RT-PCR. Here, the synergistic effect of IFN-γ with LPS to induce high expression of Nos2 and TNFα was observed while M0 genes (TBP and Adgre1) remained stable with time. Nos2, previously reported as an early expression marker showed late expression, thus, likely to be in a cell context fashion or is concentration dependent. I performed ribosome profiling coupled with RNA-seq to study global gene translation and identified LPS or IFN-γ + LPS specific genes whose expression regulations skew macrophages to M1 phenotypic state. These includes; Dusp1, Dusp2, TNF, Nos2, Socs3, Sele, NFKB, Csf2, Lyc6c2, IL6, IL10, CD83, CD86, Saa2, Egr1, Cxcl3, Cxcl10, Cxcl11 and Marco. Inducted genes were contrast in their abundance in RNA (transcriptome) compared to RPF (translatome) supporting the notion that mRNA level does not always reflect protein level. The upregulation of mitochondrial and cytosolic ribosomes coding genes identified suggested a translational level change at 1 hr and transcriptional change at 24 hrs time point. I found that IFN-γ + LPS and LPS may also modulate mRNA translation in macrophages by the enrichment of translation initiation (EIF2 signaling mTOR signaling, and regulation of eIF4 and p70S6K signaling), NF-KB pathway, Toll-like Receptor signaling and PPAR pathway which are unique pathways contributing to M1 phenotypic state and inflammatory response. Despite the difference in terms of activation and induced genes, altogether, this study provides insights into how IFN-γ and /or LPS can establish a crosstalk at the transcriptional and translational levels to achieve the differential regulation of gene sets with distinct and opposing functions.