TAMs are considered critical perpetuators of the low grade inflammation that is required to maintain tumor survival. The a2NTD peptide has been shown to stimulate monocytes to produce pro-inflammatory cytokines.17,18
In addition, a2NTD induces the expression of cytokines linked to M2 TAMs, which help in preventing immune cells from recognizing tumors.
To further characterize the interaction between monocytes and a2NTD, we are interested in determining the receptor(s) that interacts with a2NTD. While a2NTD binds to the cell surface and enters both monocytes and THP-1 cells, the former bind higher amounts of a2NTD and internalize the peptide at a much faster pace than the latter (
and Fig. S2A)
. This would suggest that THP-1 cells may express reduced amounts of the proteins that are necessary for a2NTD binding and internalization.
Our previous studies have shown that a2NTD stimulates both monocytes and THP-1 cells to produce IL-1β. However, only monocytes (but not THP-1 cells) generate reactive oxygen species (ROS) upon a2NTD stimulation.16
This is consistent with the concept that THP-1 cells express less or lack some of the proteins that mediate the endocytosis of a2NTD. Monocytes stimulated with a2NTD in the presence of trafficking inhibitors produced pro-inflammatory cytokines, indicating that the entry of a2NTD is not necessary for cytokine production ()
. Taken together, these data suggest a possible dual receptor process. Thus, while both monocytes and THP-1 cells appear to express the a2NTD receptor that is important for IL-1β secretion, only monocytes have a second receptor that brings a2NTD into the cell and leads to intracellular ROS production. In monocytes, ROS may be a redundant amplification signal to produce additional inflammatory cytokines. THP-1 cells seem not to express this putative receptor to sufficient levels and thus cannot produce ROS in response to a2NTD.
The inhibition of a2NTD endocytosis with polyI ()
indicates that a scavenger receptor is involved in a2NTD internalization. Scavenger receptors are a broad family of integral membrane proteins that mediate the cellular binding and internalization of an extraordinarily wide range of negatively charged macromolecules including lipoproteins,34
polyribonucleotides (such as polyI)35
and advanced-glycation end products.36
Macrophage have been well characterized in their expression of scavenger receptors, especially linked to their pathological role in atherosclerotic plaques.37
The a2NTD peptide carries a net negative charge at physiological pH. In addition, the a2 N-terminal domain exhibit physicochemical properties that are compatible with its integration in membranes.33
Hence, it is not surprising that scavenger receptors would mediate the endocytosis of a2NTD.
monocytes have been shown to be generally more phagocytic than their CD14+
Here, we demonstrate a preferential uptake of a2NTD by CD14++
, suggesting that a relatively specific process is taking place as opposed to unspecific phagocytosis. We have previously implicated a2NTD in promoting the development of a TAM phenotype and CD14+
monocytes are thought to constitute macrophage precursors.15
This is another piece of evidence indicating the internalization of a2NTD is a specific receptor-mediated process.
An interesting finding is the characterization of the monocyte subsets that produce cytokines in response to a2NTD stimulation. CD14+
monocytes have previously been characterized as the main producers of pro-inflammatory cytokines39,40
in an LPS stimulation model. However, both CD14++
monocytes produce significant amounts of IL-1 in response to a2NTD ()
monocytes are also a major source of TNFα upon LPS stimulation.41
In contrast, there is no significant TNFα production after the administration of a2NTD to these cells. Previous data indicate that the production of IL-1 is privileged over that of TNFα after a2NTD stimulation. The data presented here are consistent with these findings. While CD14+
monocytes have been extensively studied for their role in infectious diseases and inflammation,42
their role in tumorigenesis has only recently been explored.43,44
The interaction of CD14++
monocytes with a2NTD, stimulating the production of pro-inflammatory cytokines represents a new area of research for how the monocyte subsets are functionally defined.
In summary, the a2NTD peptide may play a prominent role in the tumor microenvironment. While functioning to promote a localized pro-inflammatory reaction, a2NTD also prevents immune surveillance, and the interaction of a2NTD with specific monocyte subsets appears to stimulate their pro-tumorigenic functions. Our data indicate there is more than one cell type responding to a2NTD. Identifying the receptor(s) of a2NTD as well as understanding the differences among different types of a2NTD-sensitive cells will perhaps drive the development of novel anticancer therapies.