Toll like receptor 9 (TLR9) is an innate immune receptor important for recognizing DNA, both of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9471-1032). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. Here we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature- or negative regulatory-form. We show that TLR9471-1032, corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by co-expression of the N-terminal fragment (TLR91-440), inclusion of the hinge region (TLR9441-1032) or overexpression of UNC93B1, the latter of which is critical for trafficking and cleavage of TLR9. TLR91-440 co-immunoprecipitates with full-length TLR9 and TLR9471-1032, but does not rescue the native glycosylation pattern, thus inappropriate trafficking likely explains why TLR9471-1032 is nonfunctional. Lastly, we show that TLR9471-1032 is also a dominant negative regulator of TLR9 signaling. Together these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.

Macrophages participate in immunity, tissue repair, and tissue homeostasis, and also mediate pathologic conditions including atherosclerosis and cancer. Depending on microenvironmental cues, macrophages may adopt a pro‐inflammatory or anti‐inflammatory phenotype. Tissue stiffness influences the behavior and differentiation of cells, yet little is known about how the physical extracellular environment influences macrophage function. To study the role of substrate stiffness in determining macrophage phenotype, we cultured murine macrophages on tunable polyacrylamide gels that approximate different tissue stiffnesses. Macrophages on rigid surfaces had larger surface area and reduced circularity compared to macrophages on compliant gels. On rigid surfaces, phagocytosis was increased, but lipid uptake was decreased. Cytokine secretion differed in macrophages grown on compliant versus rigid surfaces. Differences in response to the toll‐like receptor ligands CpG oligonucleotide and lipopolysaccharide were also noted. The distribution of vinculin, a component of mature focal adhesions that mediate mechanosensing, was punctate in macrophages grown on glass and diffuse in macrophages grown on compliant gels, suggesting a fundamental difference in the quantity and/or character of the focal adhesions. Together, our studies demonstrate that the mechanical properties of the extracellular environment have a direct impact on macrophage behavior and function. Furthermore, these findings have important implications for a variety of pathologic conditions that involve both macrophages and altered tissue stiffness such as fibrosis, atherosclerosis, and neoplasia.The studies were supported by NIH T32 Comparative Medicine Training Grant.

Atherosclerosis is characterized by the accumulation of lipid-laden macrophage foam cells within the vessel wall, formation of a plaque, and changes in tissue stiffness from soft near the necrotic core to stiff near the fibrous cap. The mechanisms governing foam cell formation are not well understood, but recent studies demonstrate that cells probe and respond to the physical properties of their surroundings. Stiffness regulates differentiation and function of mesenchymal stem cells and endothelial cells, yet until recently, its role in immune cell function has been largely unexplored. Our lab, and others, has found that substrate stiffness regulates murine macrophage cytokine secretion and phagocytic activity. Because macrophages accumulate near the soft necrotic core where there is abundant lipid, we hypothesized that macrophages on softer surfaces would exhibit enhanced lipid accumulation. To investigate this, we cultured murine macrophages on functionalized tunable polyacrylamide gels that model physiologically relevant stiffnesses. We then used a combination of flow cytometry and immunofluorescence microscopy to show that softer substrate stiffness results in more accumulation of the model lipid, oleic acid, as well as acetylated low-density lipoprotein in macrophages. Transcriptomic analysis identified up- or down-regulated genes in macrophages in response to increased substrate stiffness, including genes implicated in atherosclerosis and lipid metabolism. Together, these data demonstrate that lipid accumulation by macrophages is regulated in part by substrate stiffness and mechanotransduction signaling and reveal a potential link between changes in tissue stiffness and macrophage lipid accumulation.