High salt promotes NADPH oxidase‐dependent formation and activation of dendritic cells

It has become apparent that sodium can accumulate in the interstitium and promote inflammation through poorly defined mechanisms. We have previously shown that increased NADPH oxidase‐ dependent oxidative stress in dendritic cells (DCs) leads to formation of isolevuglandin (IsoLG)‐ modified proteins which act as neo‐antigens to activate T cells. We hypothesized that increasing sodium chloride (NaCl) in excess of the physiological interstitial space concentrations leads to monocyte differentiation into DCs in an NADPH oxidase‐dependent manner and formation of immunogenic IsoLGs. We exposed murine bone marrow cells to normal physiological NaCl (NS: 150 mM/L), elevated NaCl concentrations (HS: 190 mM/L), or HS plus gp91dstat, an inhibitor of the NADPH oxidase assembly. Compared to NS, HS significantly increased the number cells expressing the DC marker CD11c (NS: 14.4 ± 7.0 vs HS: 52 ± 11.7 % p<0.001), and this was prevented by co‐treatment with gp91dstat (39.8 ± 16.0 p<0.05 vs HS). In additional experiments, we found that exposure of monocytes isolated from human volunteers to HS, but not mannitol, caused a 2‐fold increase in formation of IsoLG‐modified proteins. This was associated with an increase in activation marker CD86 (NS: 466 ± 192 vs HS: 596 ± 324 MFI p<0.05) and production of inflammatory cytokines IL‐6, IL‐β and TNF‐α. Interestingly, these cells expressed surface markers indicative of transformation to activated DCs, as evidenced by their acquisition of surface marker CD83. In additional studies, we found that monocytes exposed to high salt for 7 days acquire a DC like morphology. Moreover, using flow cytometry, we confirmed that high salt exposure causes these cells to lose the monocyte marker CD14 (NS: 41.1 ± 15.4 vs HS: 19.9 ± 6.4 MFI; p<0.05), and gain the DC marker CD209 (NS: 24.2 ± 1.0 vs HS: 49.3 ± 0.7 MFI; p<0.001). Co‐treatment with gp91dstat and scavenging of IsoLGs during high salt exposure prevented conversion of monocytes into DCs. High salt dramatically increased mRNA expression of GM‐CSF (NS: 758 ± 440.8 vs HS: 5476 ± 2268 MFI; p<0.05), IL‐4 NS: 1868 ± 560.6 vs HS: 4867 ± 1152 MFI; p<0.05) and Flt3 (NS: 2526 ± 636.8 vs HS: 10014 ± 2370 MFI; p<0.05), which are known to mediate monocyte conversion to DCs. Thus, we have defined a novel pathway whereby high NaCl concentrations lead to transformation of monocytes to DCs due to increased formation of immunogenic isoketals. These observations provide insight into how elevated sodium environments lead to an inflammatory state. Support or Funding Information This work was supported by the American Heart Association grants 16POST29090001 and 14SFRN20420046, and National Institutes of Health grants 1K01HL13049701, 1R01HL12586501, and 5R01HL03900626