IntroductionInflammation is a highly regulated physiologic response that has evolved as a mechanism to respond to infection as well as to promote healing in settings such as tissue injury. CD4 ϩ regulatory T cells (T reg ) are observed at sites of acute and chronic inflammation, 1,2 raising questions as to the molecular basis of their accumulation at these sites. Initial evidence suggested that immunosuppressive activities of T reg are diminished in the presence of inflammatory signals. 3,4 However, in several studies, the apparently opposite picture emerged, wherein T reg exposed to inflammatory signals retain potent suppressive activity. For instance, murine T reg at sites of viral infection or isolated from inflamed tissues still mediate regulatory function, 5-7 as do human T reg isolated from rheumatoid joints or inflamed colonic mucosa. 8,9 Collectively, these results point to the possibility that certain signals associated with inflammation might promote T reg activity. Here we unexpectedly identified serum amyloid A (SAA), an acute indicator of inflammation, as a novel factor that induces cellular and cytokine conditions to support the expansion of T reg while maintaining their suppressive capacity.
MethodsIn vivo effects of SAA on T reg proliferation C57BL6/J mice (male, 8-10 weeks old) were purchased from Jackson Laboratory. Mice were injected intraperitoneally with recombinant human SAA (Peprotech, 30 g in 100 l PBS), purified human serum albumin (Sigma-Aldrich, 30 g in 100 l PBS), or endotoxin (Sigma-Aldrich, 0.25 ng in 100 l PBS). Animals were killed 16 hours later; peritoneal cells were harvested and stained for surface and intracellular markers to detect T reg frequency and proliferation. In vivo depletion of monocytes was performed with clodronate liposomes (Encapsula). In these experiments, 400 L of clodronate or empty liposomes were injected intraperitoneally 24 hours before SAA injection.
Flow cytometry and ELISADetection of surface markers and intracellular molecules was performed. Antibodies to mouse and human proteins used in these experiments are purchased from Biolegend except for anti-human formyl peptide receptor like-1 (FPRL-1; R&D Systems), anti-human RAGE (receptor of advanced glycation end products), supressor of cytokine signaling 3 (SOCS3; Abcam), and anti-Ki-67, anti-human-pAKT (phosphonylated protein kinase B), pERK1/2 (phosphorylated extracellular signal regulated kinases 1 and 2; BD Biosciences). For in vitro experiments, the relevant subset was labeled with CFSE before suppression assays. Cells were pelleted at various time points and underwent standard staining protocols of the manufacturers. For in vivo experiments, cells were harvested from peritoneal cavity and underwent flow cytometric analysis.To detect cytokines in the plasma, cytometric bead arrays (BD Biosciences) and ELISA (R&D Systems) were used according to manufacturers' protocols.
Human plasma preparationThe study was approved by the Institutional Review Board at Stanford University. All subjects provided inform...