Since the discovery of Treg much research has focused on the mechanisms involved in their development and suppression of adaptive immune responses. The influence of Treg on non-hematopoietic cells, however, has not been well studied. Although it is clear that both effector T cells and Treg migrate to sites of inflammation, there is little known about the direct regulatory effect of Treg on inflamed tissue. Here we investigated adhesive interactions between Treg and endothelium both in vitro and in vivo, and examined whether iTreg could suppress EC activation and leukocyte recruitment during acute inflammation.
Because of the widely reported presence of Foxp3+
cells in inflamed tissues of both human and mouse (10
), we hypothesized that Foxp3+
Treg may have direct anti-inflammatory effects on endothelial function. Although sub-populations of “memory like” αE
-positive Treg were previously suggested to possess high CD62P/E binding abilities (41
), the defining migratory characteristics of these Treg are unclear. We used an in vitro
flow assay to establish that Foxp3+
iTreg but not nTreg adhere and transmigrate efficiently across TNFα stimulated endothelial monolayers under physiological shear flow conditions. These results led us to believe that Foxp3+
iTreg were more likely to successfully migrate to sites of inflammation and were therefore more relevant to our study. Moreover, antigenic activation of Foxp3+
iTreg in the proximity of endothelial cells triggers a regulatory response that rapidly suppresses endothelial activation by TNFα and IL-1β, as evidenced by a decrease in endothelial selectin expression and effector T cell adhesion. We further demonstrated that this suppressive effect is contact independent and principally mediated by the TGFβ/ALK5 signaling pathway in EC.
Endothelial cell responses to TGFβ are complex because they have two distinct signaling pathways that lead to different and competing outcomes. TGFβ signaling is initiated when the cytokine brings together two receptors, TGFβ type-II receptor (TβRII) and TGFβ type-I receptor (TβRI) (also known as ALK5). In most types of cells, the formation of the heteromeric complex TβRII-TGFβ-TβRI allows the constitutively active kinase in the cytoplasmic tail of TβRII to phosphorylate/activate the cytoplasmic tail of TβRI/ALK5, which in turn activates the Smad2/3 cascades (38
). In addition to TβRII and TβRI, endothelial cells express endoglin (CD105) that joins into a complex with surface TβRII and diverts it to signal preferentially through the ALK-1/Smad1/5/8 cascade (43
). The ALK1/Smad1/5/8 pathway promotes endothelial cell proliferation and migration and is inhibited by signaling from the ALK5/Smad2/3 pathway (45
). Moreover, endoglin expression is important for endothelial cell survival in the presence of TGFβ1 (46
) and endoglin deficiency is the cause for vascular dysplasia and reoccurring hemorrhages that underlie hereditary hemorrhagic telangiectasia type 1 (HHT-1) (47
We found that MHEC expressed significant levels of ALK1, ALK5 and endoglin mRNA and that expression levels were not significantly affected by exposure to iTreg supernatant or to TGFβ1 (Supplementary Figure S4 A,B,C
). Notably, the levels of endoglin mRNA were very high (33±1.6% of Actb expression) and surface endoglin was high and remained unchanged after stimulation with TNFα (Supplementary Figure S4 D
). It was recently suggested that TNFα induces endoglin shedding in cultured human umbilical vain endothelial cells (HUVEC) and that TNFα is an important mediator of the elevation of soluble endoglin (sEng) during preeclampsia (48
). Nonetheless, the current study demonstrates that ALK5 signaling has a dominant role in Foxp3+
iTreg suppression despite high levels of surface endoglin on endothelial cells.
Another possible explanation for the suppressive effects of iTreg supernatant in our study is that soluble TNF receptor-II (sTNFRII, sCD120b) that is shed by activated Treg (49
) blocked the soluble recombinant TNFα we used to stimulate endothelial cells. There are several reasons, however, why this explanation is unlikely. First, it is established that sTNFRII binds mainly to membrane-bound TNFα and has very low binding capacity for soluble TNFα (50
). Second, throughout our study, we stimulated MHEC with soluble TNFα at a range of concentrations (25–100ng/mL) that are 5–20 fold higher than the levels of sTNFRII reported in supernatants of activated Treg of both murine and human (5–6ng/mL) (49
). Third, in experiments where we used IL-1β to stimulate the endothelium, we found no reduction in iTreg capacity to suppress selectin expression and leukocyte recruitment. Lastly, the suppressive effect of iTreg supernatant was not affected following removal of sTNFRII via immune-precipitation with magnetic beads labeled with goat anti-mouse sTNFRII capture IgG (data not shown).
The differences in activation status between nTreg and iTreg may underlie some of the observed differences in adhesion and transmigration behavior. iTreg are rapidly formed from activated naïve T cells, proliferate as long as they are provided IL-2 and then disappear without leaving a known ‘rested’ memory cells. On the other hand, only minute fraction of peripheral nTreg are recent thymus emigrants with an activated phenotype. In order to test if recent activation promotes nTreg adhesion we stimulated purified nTreg with either plate bond stimulating anti- CD3ε antibody or with spleen derived antigen presenting cells in combination with stimulating anti- CD3ε antibody and tested adhesion after various time points. These experiments, that we performed as part of our preliminary studies, have demonstrated that TCR activation does not change the adhesion and transmigration behavior of nTreg. Moreover the addition of cytokines such as IL-2 and TGFβ1 and supporting co-stimulation with agonistic anti-CD28 antibody did not change these outcomes and again re-stimulated nTreg showed very poor interactions with cytokine stimulated EC or with immobilized selectin ligand (data not shown).
The current study suggests that Foxp3+
Treg that form naturally in response to non pathological oral and intranasal antigens (9
) interact with inflamed endothelium and suppress further endothelial activation and leukocyte recruitment. This view is complementary to a recent study by Clark and colleagues (52
) which demonstrates that nTreg but not iTreg require TNFα or re-activation in the presence of exogenous TGFβ1 to suppress CD4+
T cell mediated colitis in the recombinant activating gene (RAG) deficient mice. This study supports our observation that TGFβ1 released by re-activated iTreg () but not nTreg (data not shown) mediated swift inhibition of endothelial activation.
The presence of Foxp3+ Treg at inflammatory sites in humans and mouse models has led to speculation about their possible function as direct suppressors of tissue inflammation. Although the ability to interfere with antigen presentation is likely to be one important function of Treg, the suppressive effect of Treg have on non-hematopoeitic cells during inflammation may be under appreciated. Because the endothelium is a major target for effector T-cell derived pro-inflammatory cytokines, it is plausible that Treg influence EC in order to control the spread and pathological side effects of inflammation. Nevertheless, it is technically intricate to isolate Treg influence on endothelium in vivo. In the current work we demonstrated that iTreg strongly interact with cytokine stimulated endothelial monolayers in vitro and are capable of suppressing adhesive interactions with effector T cells. iTreg suppression was mediated by TGFβ in an ALK5 dependent manner and was triggered either in response to antigenic stimulus by a third party which we experimentally demonstrated by carrying over supernatant, or by direct contact and immune synapse formation with endothelial antigen presentation. The in vivo relevance of these findings was confirmed first by intravital microscopy of iTreg rolling on inflamed endothelium, and then by demonstrating the robust suppressive capacity of iTreg secretion products on leukocyte recruitment during acute peritonitis. Taken together, our data suggests that Foxp3+ iTreg are capable of controlling inflammation through direct suppression of endothelial activation and leukocyte recruitment in a manner independent of their influence on effector T cell activation and proliferation.