Molecular Mechanism And Function Of Grass Carp Transforming Growth Factor-β1in Teleost Immunity

TGF-β1belongs to the TGF-β1superfamily, and its pivotal regulatory roles inmammalian immune system have already been extensively characterized. Generally,TGF-β1promotes the expression of Foxp3in activated na ve CD4+cells, resulting inthe generation of the Foxp3+Treg subgroup, which acts as a potent immune suppressivemediator to maintain the immune tolerance and prevent autoimmunity.However, the functional role of TGF-β1in the immune system of teleost is poorlyunderstood. In this study, we examined the immunoregulatory effects of TGF-β1ingrass carp peripheral blood leukocytes (PBL) and head kidney leukocytes (HKL). It isinteresting that TGF-β1consistently stimulated the cell viability and the mRNA levelsof pro-inflammatory cytokines (tnfα and ifnγ) and T/B cell markers [cd4-like (cd4l),cd8α, cd8β and igμ] in PBL, which contrasted with its inhibitory tone in HKL. It seemsthat the bidirectional function of TGF-β1in grass carp may correlate with the oppositestatus of the subsets in PBL and HKL.Further studies showed that grass carp TGF-β1type I receptor, activin receptor-likekinase5(ALK5), was indispensable for the immunoregulatory effects of TGF-β1inPBL and HKL. Notably, TGF-β1persistently attenuated ALK5expression, whereasimmunoneutralization of endogenous grass carp TGF-β1could increase ALK5mRNAand protein levels. It is consistent with the observation that TGF-β1decreased thenumber of ALK5+leukocytes in PBL and HKL, revealing a negative regulation ofTGF-β1signaling at the receptor level. Moreover, transient treatment with TGF-β1for24h was sufficient to induce similar cellular responses compared with the continuoustreatment. This indicated a possible mechanism by which TGF-β1triggered thedown-regulation of ALK5mRNA and protein, leading to the desensitization of grasscarp leukocytes toward TGF-β1.Considering the potential effect of TGF-β1on Foxp3expression and the crucialrole of Foxp3expression in Treg activity in mammals, grass carp foxp3(gcfoxp3) genewas cloned and identified in this study. As expected, TGF-β1was effective in up-regulating gcFoxp3mRNA and protein levels in HKL. Meanwhile, TGF-β1inducedthe generation of grass carp Foxp3+cells. Similar to mammalian Treg, these cellsdisplayed immune suppressive activities in co-culture system in which Foxp3+cellsinduced by TGF-β1down-regulated the cell viability and the expression of effectormolecules (tnfα and ifnγ) of the responder cells stimulated by PHA. A possibleexplanation for the suppressive functions of these Foxp3+cells could be due to thesecretion of cytokines (TGF-β1and IL-10) via a non-cell contact mechanism.Accordingly, our data revealed a dual role of TGF-β1in teleost immunity in whichit can serve as a positive or negative control device and provided additional mechanisticinsights as to how TGF-β1controls its signaling in vertebrate leukocytes. Furthermore,we demonstrated the regulatory mechanism of 'TGF-β1-Foxp3-Treg-like cells' axis inteleost.