Epithelial sodium channels in macrophage migration and polarization: role of proinflammatory cytokines TNFα and IFNγ.

Migration of monocytes-macrophages plays an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. Although epithelial Na+ channels (ENaCs) are required for normal migratory responses in other cell types, their role in macrophage migration signaling is unknown. To address this possibility, we determined whether ENaC message is present in several peripheral blood monocyte cell populations and tissue-resident macrophages in healthy humans using the Human Protein Atlas database (www.proteinatlas.org) and the mouse monocyte cell line RAW 264.7 using RT-PCR. We then determined that selective ENaC inhibition with amiloride inhibited chemotactic migration (∼50%), but not phagocytosis, of the mouse monocyte-macrophage cell line RAW 264.7. Furthermore, we generated a cell line stably expressing an NH2-terminal truncated αENaC to interrupt normal channel trafficking and found it suppressed migration. Prolonged exposure (48 h) of RAW 264.7 cells to proinflammatory cytokines interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) inhibited RAW 264.7 migration and abolished the amiloride (1 µM)-sensitive component of migration, a finding consistent with ENaC downregulation. To determine if proinflammatory cytokines regulate αENaC protein expression, cells were exposed to proinflammatory cytokines IFNγ (10 ng/mL, last 48 h) and TNFα (10 ng/mL, last 24 h). By Western blot analysis, we found whole cell αENaC protein is reduced ≥50%. Immunofluorescence demonstrated heterogeneous αENaC inhibition. Finally, we found that overnight exposure to amiloride stimulated morphological changes and increased polarization marker expression. Our findings suggest that ENaC may be a critical molecule in macrophage migration and polarization.

Bone marrow monocytes and macrophages from mice lacking ßENaC and ASIC2 have a reduced chemotactic migration response and polarization.

The monocyte-macrophage system plays an important role in phagocytosis of pathogens and cellular debris following infection or tissue injury in several pathophysiological conditions. We examined ENaC/ASIC subunit transcript expression and the importance of select subunits in migration of bone marrow derived monocytes (freshly isolated) and macrophages (monocytes differentiated in culture). We also examined the effect of select subunit deletion on macrophage phenotype. BM monocytes were harvested from the femurs of male and female WT and KO mice (6-12 weeks of age). Our results show that α, ß, γENaC, and ASIC1-5 transcripts are expressed in BM macrophages and monocytes to varying degrees. At least αENaC, ßENaC, and ASIC2 subunits contribute to chemotactic migration responses in BM monocyte-macrophages. Polarization markers (CD86, soluble TNFα) in BM macrophages from mice lacking ASIC2a plus ßENaC were shifted towards the M1 phenotype. Furthermore, select M1 phenotypic markers were recovered with rescue of ßENaC or ASIC2. Taken together, these data suggest that ßENaC and ASIC2 play an important role in BM macrophage migration and loss of ßENaC and/or ASIC2 partially polarizes macrophages to the M1 phenotype. Thus, targeting ENaC/ASIC expression in BM macrophages may regulate their ability to migrate to sites of injury.