The immunosuppressive status of the tumor microenvironment (TME) remains poorly defined due to a lack of understanding regarding the function of tumor-associated macrophages (TAMs), which are abundant in the TME. TAMs are crucial drivers of tumor progression, metastasis, and resistance to therapy. Intra- and inter-tumoral spatial heterogeneities are potential keys to understanding the relationships between subpopulations of TAMs and their functions. Antitumor M1-like and pro-tumor M2-like TAMs coexist within tumors, and the opposing effects of these M1/M2 subpopulations on tumors directly impact current strategies to improve antitumor immune responses. Recent studies have found significant differences among monocytes or macrophages from distinct tumors, and other investigations have explored the existence of diverse TAM subsets at the molecular level. In this review, we discuss emerging evidence highlighting the redefinition of TAM subpopulations and functions in the TME and the possibility of separating macrophage subsets with distinct functions into antitumor M1-like and pro-tumor M2-like TAMs during the development of tumors. Such redefinition may relate to the differential cellular origin and monocyte and macrophage plasticity or heterogeneity of TAMs, which all potentially impact macrophage biomarkers and our understanding of how the phenotypes of TAMs are dictated by their ontogeny, activation status, and localization. Therefore, the detailed landscape of TAMs must be deciphered with the integration of new technologies, such as multiplexed immunohistochemistry (mIHC), mass cytometry by time-of-flight (CyTOF), single-cell RNA-seq (scRNA-seq), spatial transcriptomics, and systems biology approaches, for analyses of the TME.

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function.

Fine-tuning of inflammatory responses by microRNAs (miRNAs) is complex, as they can both enhance and repress expression of pro-inflammatory mediators. In this study, we investigate inflammatory responses following global miRNA depletion, to better define the overall contribution of miRNAs to inflammation. We demonstrate that miRNAs positively regulate Toll-like receptor signaling using inducible Dicer1 deletion and global miRNA depletion. We establish an important contribution of miR-19b in this effect, which potentiates nuclear factor-κB (NF-κB) activity in human and mouse cells. Positive regulation of NF-κB signaling by miR-19b involves the coordinated suppression of a regulon of negative regulators of NF-κB signaling (including A20/Tnfaip3, Rnf11, Fbxl11/Kdm2a and Zbtb16). Transfection of miR-19b mimics exacerbated the inflammatory activation of rheumatoid arthritis primary fibroblast-like synoviocytes, demonstrating its physiological importance in the pathology of this disease. This study constitutes, to our knowledge, the first description of a miR-19 regulon that controls NF-κB signaling, and suggests that targeting this miRNA and linked family members could regulate the activity of NF-κB signaling in inflammation.

Human TLR7 and 8 (hTLR7/8) have been implicated in the sequence-dependent detection of RNA oligonucleotides in immune cells. Although hTLR7 sequence-specific sensing of short RNAs has been inferred from studies of murine TLR7, this has yet to be established for hTLR7. We found that different short ssRNA sequences selectively induced either TNF-α or IFN-α in human PBMCs. The sequence-specific TNF-α response to ssRNAs observed in PBMCs could be replicated in activated human macrophage-like (THP-1) cells pretreated with IFN-γ. Surprisingly, suppression of hTLR7 expression by RNA interference in this model reduced sensing of all immunostimulatory ssRNAs tested. Modulation of the relative expression ratio of hTLR7 to hTLR8 in THP-1 cells correlated with differential sensing of immunostimulatory sequences. Furthermore, the sequence-specific IFN-α induction profile in human PBMCs was accurately modeled by a sequence-specific activation of murine TLR7 in mouse macrophages. Thus, we demonstrate for the first time that hTLR7 is involved in sequence-specific sensing of ssRNAs. We establish a novel cell model for the prediction of TNF-α induction by short RNAs in human macrophages. Our results suggest that differential sequence-specific sensing of RNA oligonucleotides between human and mouse macrophages is due to the modulation of TLR7 sensing by human TLR8.

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