Smad3-Deficient CD11b+Gr1+ Myeloid-Derived Suppressor Cells Prevent Allograft Rejection via the Nitric Oxide Pathway

Abstract

Immunosuppressive CD11b+Gr1+ myeloid-derived suppressor cells and TGF-β have been shown to negatively regulate host immunity against allografts. Our results demonstrated that Smad3-deficient mice or mice reconstituted with Smad3-deficient hematopoietic cells rejected allogeneic skin or heart grafts in a significantly slower manner compared with littermates or wild-type (WT) control mice. Transplanted Smad3−/− recipients produced markedly less anti-donor IgG Abs, especially IgG1 and IgG2b subclasses. T cells in alloskin-grafted Smad3-deficient mice were more likely to participate in a Th2-type immune response, as evidenced by more Th2-specific transcription factor, GATA3 expression, and increased IL-4 and IL-10 production, as well as less Th1-specific transcription factor, T-bet expression, and decreased IL-2 and IFN-γ production. More CD11b+Gr1+ neutrophil infiltration and less monocyte/macrophage and T cell infiltration in allografts were observed in Smad3−/− recipients compared with WT recipients. Increased CXCL1 and CXCL2 as well as decreased CCL3, MCP-1, and RANTES chemokines in allografts of Smad3−/− recipients were consistently detected by real-time PCR. Further studies indicated that the increased CD11b+Gr1+ myeloid cells in Smad3-deficient mice were immunosuppressive and responsible for the delayed allograft rejection mainly via an NO-dependent pathway. Thus, this study identifies Smad3 as an intrinsic negative regulator that critically inhibits the differentiation and function of immunosuppressive CD11b+Gr1+ myeloid-derived suppressor cells.

TSC1 controls macrophage polarization to prevent inflammatory disease

“…Bone marrow cells were cultured with Dulbecco's modified Eagle medium (DMEM) containing 10% (v/v) FBS and 10 ng ml À 1 of mouse M-CSF or 10 ng ml À 1 of mouse GM-CSF for 12 days to obtain bone marrowderived macrophagess 56 . Primary mouse peritoneal macrophages were obtained from the peritoneal exudates of 4-to-6-week-old mice 57 . The peritoneal exudate cells were washed twice with PBS solution and adjusted to 1 Â 10 6 cells ml À 1 in DMEM cultured for 3-4 h at 37°C and 5% CO 2 .…”

TSC1 controls IL-1β expression in macrophages via mTORC1-dependent C/EBPβ pathway

“…Total RNA was isolated with TRIzol (Invitrogen), and reverse transcription was performed with M-MLV superscript reverse transcriptase according to the manufacturer's instructions 32 . Real-time PCR kit (SYBR Premix Ex Taq, DRR041A) was purchased from Takara Bio Inc. PCR was performed on CFX96 (Bio-Rad).…”

Targeting S1P1 Receptor Protects against Murine Immunological Hepatic Injury through Myeloid-Derived Suppressor Cells

“…The expression of each target gene is presented as the fold change relative to that of control samples (2 -DDCT ), as described previously (33). Immunoblot analysis was done, as described (34) (38). Purified T cells were plated at a density of 2 3 10 5 cells/well in 1 mg/ml anti-CD3 Ab-coated plates in the presence of 1 mg/ml anti-CD28 Ab.…”

“…After incubating equal volumes of culture supernatant or serum (100 ml) with Greiss reagent (G4410; Sigma-Aldrich), the absorbance at 550 nm was measured using a microplate reader (Bio-Rad, Hercules, CA), as described (38). The concentrations of serum CXCL1 and CXCL2 and serum or culture supernatant IFN-g were quantified by sandwich ELISA (MKC00B, MM200, and MIF00; R&D Systems).…”

“…3C) and spleen (data not shown) displayed significantly enhanced immunosuppressive activities on CD4 + T cell proliferation in vitro. We further depleted these cells by injecting anti-Gr1 mAb, as described previously (38). Of note, an efficient depletion of CD11b + Gr1…”