Functions of Smad Transcription Factors in TGF-ß1-Induced Selectin Ligand Expression on Murine CD4 Th Cells.

Selectins are carbohydrate-binding adhesion molecules that control leukocyte traffic. Induction of selectin ligands on T cells is controlled primarily by cytokines, including TGF-ß1, and requires p38α MAPK, but transcriptional mechanisms that underlie cytokine-driven selectin ligand expression are poorly understood. In this study, we show, using mice with conditional deletions of the TGF-ß1-responsive transcription factors Smad2, Smad3, or Smad4, that induction of selectin ligands on CD4 cells in response to TGF-ß1 requires Smad4 plus either Smad2 or Smad3. Analysis of CD4 cells from mice with only one functional Smad4 allele revealed a sharp gene dosage effect, suggesting the existence of a threshold of TGF-ß1 signal strength required for selectin ligand induction. Both Smad4 plus either Smad2 or Smad3 were selectively required for induction of Fut7 and Gcnt1, glycosyltransferases critical for selectin ligand biosynthesis, but they were not required for St3gal4 or St3gal6 induction. Smad4 plus either Smad2 or Smad3 were also required for induction of Runx transcription factors by TGF-ß1. Enforced expression of Runx2, but not Runx1 or Runx3, in Smad2/Smad3 doubly deficient CD4 cells restored selectin ligand expression to wild-type levels. In contrast, enforced expression of Runx1, Runx2, or Runx3 failed to restore differentiation of TGF-ß1-dependent Th cell lineages, including Th17, Th9, and induced regulatory T cells. These results show that Smads are directly required for Th cell differentiation independent of Runx induction but only indirectly required via Runx2 for TGF-ß1-induced selectin ligand induction on murine CD4 T cells.

DHA-enriched fish oil targets B cell lipid microdomains and enhances ex vivo and in vivo B cell function.

DHA is a n-3 LCPUFA in fish oil that generally suppresses T lymphocyte function. However, the effect of fish oil on B cell function remains relatively understudied. Given the important role of B cells in gut immunity and increasing human fish oil supplementation, we sought to determine whether DFO leads to enhanced B cell activation in the SMAD-/- colitis-prone mouse model, similar to that observed with C57BL/6 mice. This study tested the hypothesis that DHA from fish oil is incorporated into the B cell membrane to alter lipid microdomain clustering and enhance B cell function. Purified, splenic B cells from DFO-fed mice displayed increased DHA levels and diminished GM1 microdomain clustering. DFO enhanced LPS-induced B cell secretion of IL-6 and TNF-α and increased CD40 expression ex vivo compared with CON. Despite increased MHCII expression in the unstimulated ex vivo B cells from DFO-fed mice, we observed no difference in ex vivo OVA-FITC uptake in B cells from DFO or CON mice. In vivo, DFO increased lymphoid tissue B cell populations and surface markers of activation compared with CON. Finally, we investigated whether these ex vivo and in vivo observations were consistent with systemic changes. Indeed, DFO-fed mice had significantly higher plasma IL-5, IL-13, and IL-9 (Th2-biasing cytokines) and cecal IgA compared with CON. These results support the hypothesis and an emerging concept that fish oil enhances B cell function in vivo.