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The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg as well as their direct influence at the site of inflammation remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3+ adaptive/inducible Treg (iTreg) but not naturally occurring Treg (nTreg) efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial antigen presentation or immobilized anti-CD3ε, Foxp3+ iTreg suppressed TNFα and IL-1β mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGFβ-induced activin receptor like kinase [ALK]5 signaling in endothelial cells. In addition, Foxp3+ iTreg adhered to inflamed endothelium in vivo and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3+ iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.
Treg have a critical role in protecting against autoimmunity and regulating immune responses by suppressing T cell proliferation (1). The ability of Treg to migrate to sites of inflammation is an important factor for their suppressive function (2), however their direct influence at the site of inflammation is still unknown. The two main types of Treg are naturally occurring regulatory T cells (nTreg) and adaptive/inducible Treg (iTreg). nTreg develop in the thymus, where they acquire expression of the transcription factor Foxp3 in response to self antigen presentation. Fully mature nTreg then migrate to the secondary lymphoid organs, where they suppress the expansion of self reactive T cells (3). In contrast, Foxp3+ iTreg develop from naive CD4+ T cells in the lymphoid tissues in response to environmental antigens presented in association with TGFβ1 but without the influence of “danger signals,” such as pathogen- or damage-associated molecular patterns (PAMPs and DAMPs), or inflammatory cytokines that stimulate antigen presenting cells (APCs), such as IFNγ, IL-1, IL-6 and IL-12 (4). Antigen presentation in the absence of these products, often referred to as ‘tolerogenic’, occurs with self antigens and environmental antigens collected by APCs around the mucosal lining of the nasal passages, the lung, and the gut. Tolerogenic antigen presentation is a central mechanism in suppressing undesired immune reactivity against non harmful material such as airborne particles, food and commensal bacteria. In vitro studies show that TGFβ1 and IL-2 are critical factors for developing Foxp3+ iTreg (5) and that IL-4 and IFNγ antagonize the acquisition of Foxp3 expression that orchestrates these iTreg function. Moreover, the vitamin A metabolite, retinoic acid (RA), that is naturally synthesized by tolerogenic APCs, stabilizes Foxp3+ iTreg by blocking these antagonistic effects of IL-4 and IFNγ (6, 7). In addition, RA promotes TGFβ1 mediated Foxp3 expression during in vitro differentiation of iTreg and preferentially supports Foxp3+ iTreg formation over Th17 (8). Interestingly, both nTreg and iTreg seem to have similar capacities to suppress T cell proliferation in vitro when using anti-CD3ε as a TCR stimulus. Nevertheless, the overall contributions of nTreg and iTreg to immune tolerance may be quite different with respect to mechanism and antigen specificities (9). iTreg are essentially non self-reactive and respond to foreign antigenic stimulation, while nTreg have self-reactive TCR specificities that suppress autoimmune responses (4). Therefore, the presence of each Treg subset at the site of inflammation may have different implications.
Interestingly, Treg are found at the sites of inflammation in patients with various types of inflammatory conditions and in murine models of these diseases, including inflammatory bowel disease (10), lung injury (11), atherosclerosis (12), experimental autoimmune encephalomyelitis (EAE) (13) and transplantation (14). Moreover, it was demonstrated that the ability of Treg to migrate to sites of inflammation is a prerequisite for their subsequent suppressive function in the draining lymph nodes (2). We have recently shown that Treg migrate into atherosclerotic lesions and that persistent hypercholesterolemia leads to a reduction in Treg migration and an associated increase in disease progression (15). Nonetheless, in spite of growing interest in the therapeutic potential of in vitro expanded nTreg and in vitro generated iTreg (16, 17), the migratory characteristics of “inflammation visiting” Treg and the specific nature of their activity at the site of inflammation remain unclear. Here we characterize the adhesive behavior of Foxp3+ iTreg and nTreg with inflamed endothelium. We demonstrate that only Foxp3+ iTreg adhere to cytokine stimulated endothelial cells (EC) and transmigrate in vitro at similar levels to Th1 effector cells. Upon contact with endothelial cells in vitro, Foxp3+ iTreg respond to endothelial antigen-presentation and suppress TNFα and IL-1β mediated leukocyte recruitment by regulating the expression of endothelial CD62E and CD62P (E- and P-selectins accordingly). We also find that TGFβ1, which is released by Foxp3+ iTreg upon re-activation, mediates the suppressive effects of iTreg on endothelial activation in an AKL5 dependent manner. Using intravital microscopy of leukocyte rolling in the microvasculature of the mouse cremaster muscle, we also show that Foxp3+ iTreg have strong interactions with inflamed endothelium in vivo. Finally, we demonstrate that both TGFβ1 and iTreg secretion products are able to suppress leukocyte recruitment in a model of TNFα induced acute peritonitis. These data demonstrate the suppressive influences of Foxp3+ iTreg on endothelial activation and leukocyte recruitment and provide a novel insight into their regulatory function at the site of inflammation.
C57BL/6 mice referred to as ‘wild type’ used in the study were purchased from Jackson Laboratory (Bar Harbor, ME); Foxp3-eGFP knock-in mice that express cytoplasmic green fluorescence protein (GFP) as reporter for Foxp3 expression (18) were kindly provided by Dr Vijay Kuchroo (Center for Neurological Disease, Brigham and Women’s Hospital); SMARTA T cell receptor transgenic (TCR-Tg) mice, that express LCMVgp specific CD4+ T cells, were a kind gift from Dr Pamela Ohashi (19) and the SMARTA x Foxp3-eGFP mice are the crossbreed of the above two strains on the C57BL/6 background. Mice were used for experiments at 8 to 12 weeks of age in a sex match setup. Male and female mice were used in separate experiments, with no differences in response to our experimental treatments in this study. Mice were housed and bred in the pathogen-free facility at the New Research Building, Harvard Medical School. All procedures done with animals were conducted in accordance to protocols approved and supervised by the Institutional Committee for Animal Research at the Harvard Medical School in accordance with the National Institutes of Health guidelines for animal research.
nTreg were isolated from spleens of Foxp3-eGFP knock-in mice as previously described (18). Foxp3+ iTreg were derived from naive CD4+ CD25− GFP− cells purified from spleens of the SMARTA x Foxp3-eGFP mice using a combination of CD4-MACS magnetic beads and FACS sorting for GFP-cells. These cells were stimulated with plate bound anti-CD3ε (5μg/mL) in culture media with the addition of activating anti-murine CD28 (2μg/mL), human recombinant TGFβ1 (10ng/mL), IL-2 (50U/μL), anti- murine IL-4 (0.5μg/mL), anti- murine IFNγ (2μg/mL) with or without the addition of retinoic acid (RA) at concentration of 100nM. For more clarity, Foxp3+ iTreg generated in the presence of RA were marked as iTreg-RA. After 48h of activation, cells were transferred from the anti-CD3ε covered plates to a fresh tissue culture plates for another three days and then Foxp3-GFP+ iTreg were sorted by FACS and expression of Foxp3 protein was verified by intracellular staining. Both nTreg and iTreg were sorted for high purity (>98%) and tested for suppression of responder T cell proliferation (supplementary Figures S1 and S2). Similarly, Th1 were developed from naive CD4+ that were activated by plate bound anti-CD3ε with the addition of activating anti-CD28 (2μg/mL) in the presence of IL-4 blocking antibody and IL-12 (10ng/mL). Th1 phenotype was confirmed by intracellular staining of IFNγ and typically more than 95% of the viable cells were IFNγ+. Flow cytometry data was processed using FlowJo software (Tree Star Inc., Ashland OR USA).
iTreg were generated in vitro as above described, purified at day 5 of culture, then rested for one day in media containing IL-2 (50U/μL), then washed and re-activated by transfer to a 24 well culture dish (2×106 iTreg in 1mL/well) containing antigen-pulsed murine heart endothelial cells (MHEC) or plate bound anti-CD3ε. Sixteen hours later, the supernatant was collected, centrifuged and stored at −20°C. In order to block the effect of sTNFRII, supernatants were absorbed with magnetic beads coated with affinity purified goat anti-mouse TNFRII IgG (R&D Systems).
MHEC were prepared using MACS- Immunomagnetic beads (Miltenyi Biotech) specific for CD31 and ICAM-2 as previously described (20). Briefly, sheep-anti-rat-IgG Dynal beads are coated with either anti-PECAM-1 (CD31) or anti-ICAM-2 (CD102) mAb. Hearts are harvested from 2 mice per preparation, minced, and then digested in collagenase (180–200U/ml) at 37°C for 45 min. The digested tissue is filtered through a 70 μm cell strainer, washed and then incubated with PECAM-1-coated beads. The bead bound cells are magnetically recovered and cultured in DMEM containing 20% FCS, supplemented with 100 μg/ml porcine heparin, and 100μg/ml endothelial cell growth stimulant (ECGS-Biomedical Technologies) in gelatin-coated tissue culture flask. After reaching 70–80% confluence (4–6 days), cells are detached from the culture dish with trypsin-EDTA, and subjected to a second selection step using anti-ICAM-2 coated beads. The bead bound cells are then magnetically recovered. Confluent monolayers of MHEC are used for experiments at passages 1–3.
T cell adhesion to endothelial cell monolayers and trans-endothelial migration (TEM) were measured in vitro as previously described (21). Briefly MHECs at the 2–4 culture passage were plated at confluence on fibronectin-coated 25-mm glass coverslips and incubated overnight to allow monolayer formation. At the day of experiment, these endothelial monolayers on coverslips were stimulated with TNFα (5–50ng/mL) or IL-1β (5–10ng/mL) for 3–4 hours, placed in flow chamber, and adhesion trans-migration assays were performed as previously described (20). T cells were suspended (1×106/mL) in flow buffer (PBS added with 0.1% horse serum albumin DPBS/0.1% HSA) and perfused across the endothelial monolayers at 37°C under shear stresses of 0.8 or 1.0 dynes/cm2. Rolling, firm-adhesion and TEM activities were recorded by video images captured by the MetaMorph imaging system, and analyzed for number of surface accumulated cells, rolling fraction and velocities, and number of transmigrated cells, in a minimum of three high-power (40X objective) fields. To evaluate the influence of iTreg supernatant on endothelial function and CD62E and CD62P expression, MHEC monolayers were prepared as described above and sTNFRII absorbed iTreg supernatant was added simultaneously with TNFα or IL-1β used for stimulation. TGFβ receptor blockade was achieved by treating endothelial cells with the ALK5 inhibitor SB431542 hydrate (Sigma) at concentrations of 500–1000nM from 40 min before the addition of iTreg supernatant until the end of incubation (3–4h for adhesion assay and for evaluation surface CD62E and CD62P by flow cytometry or 6h for mRNA analysis). SB431542 specifically suppress TGFβ1 signaling in EC. Multiple studies have demonstrated that SB-431542 blocks ALK5, which is abundantly expressed by EC. Although SB-431542 blocks other ALK moleculesm, namely ALK4 and ALK7, these ALKs are not expressed by EC and are not related to TGF-β signaling. They preferentially form dimers with the activin type-IIB receptor (ACTR-IIB) and respond to activin ligands and not to TGFβ1(22). Furthermore, SB431542 has no effect on ALK1, -2, -3, and -6 that respond to TGFβ1 as well as to bone morphogenetic proteins (BMPs) signaling (23, 24).
Interactions of T cells and Treg with recombinant mouse E-selectin Fc- chimera-coated coverslips (R&D Systems) were examined under defined laminar flow conditions in a parallel plate flow chamber as described previously (25, 26). T cells were suspended in Dulbecco’s phosphate-buffered saline containing 0.1% (v/v) BSA and 20mM HEPES, pH 7.4, at 37°C (5 x105 cells/mL) and perfused over the coated coverslips. T cell interactions were recorded with a phase contrast objective (20x) and a video microscope connected to VideoLab software (Ed Marcus Laboratories) to record cell behavior (shear force of 0.8 and 1dyn/cm2). Accumulation of the cells was determined after the initial minute of each flow rate by counting cells in five different fields.
Intravital microscopy studies of the mouse cremaster muscle microcirculation were performed as described (27, 28). Mouse recombinant TNF-α (0.5 μg in 200μL saline) was injected intrascrotally 1.5h prior to cremaster exteriorization. Mice were anesthetized and a microcatheter was introduced into the right femoral artery to enable retrograde injection of fluorescently-labeled T cells (28). Transmitted light and fluorescent cremaster imaging was done with an Olympus FV1000 confocal intravital microscope using a 40X water immersion objective. Fluorescence imaging was done sequentially at 473nM and 635nM to reduce the potential for channel crosstalk. The centerline red blood cell velocity (Vcl) in each venule was measured in real time with an optical Doppler velocimeter (Texas A&M, College Station, TX) and Vcl was used to determine the wall shear rate and critical velocity (Vcrit) (28). GFP-iTreg and Alexa 680-labeled GFP-naïve T cells were suspended at 30×107 cells/ml and small boluses (3×106 of each cell type) of a mixture of both cells were injected retrograde into the femoral artery catheter. Rolling interactions of adoptively transferred T cells in post-capillary venules of the cremaster muscle were then visualized. Microvessel images were analyzed off-line using Imaris software (Bitplane, South Windsor, CT).
Peritonitis was induced in C57BL/6 mice by a single intra-peritoneal (i.p.) injection of TNFα (12.5ng). Four hours later mice were sacrificed and the number of leukocytes present in the peritoneal lavage fluid was evaluated by direct counts and flow cytometric analysis. To test the influence of iTreg or TGFβ1 on leukocyte recruitment, mice were pre-injected i.p. with 1mL of iTreg supernatant or 1mL of culture media with or without the addition of TGFβ1 (4ng) 30 minutes prior to the induction of peritonitis..
Plot charts and statistical analysis including Student t test for experiments with two groups or one-way ANOVA with Tukey multiple comparison post test for experiments with 3 or more groups. Statistical analysis was performed using GraphPad Prism version 5.00 for Windows, (GraphPad Software, San Diego California USA, www.graphpad.com).
We tested the main subsets of Treg, Foxp3+ nTreg and Foxp3+ iTreg, for the ability to interact with TNFα stimulated endothelial cells under shear flow conditions. In order to study the adhesion and migratory function of iTreg in a comparative manner, we used naïve T cells as a negative control and Th1 cells as a positive control, because these subsets have low and high capacities, respectively, to adhere to cytokine activated endothelium and to migrate to sites of inflammation (29). MHEC were grown in monolayers, stimulated with TNFα, and the subsequent adhesive interactions of Treg with the MHEC were measured in a flow chamber assay. In this model, Foxp3+ iTreg formed more adhesive interactions with TNFα-stimulated MHEC (64.95±1.18% of the number of Th1 interactions) as compared to nTreg and naive T cells (29.2±4.58% and 10.2±0.11% of the number of Th1 interactions, respectively) (Figure 1A). Moreover, we found large differences in the ability of Foxp3+ iTreg and nTreg to transmigrate through the MHEC monolayer. Of the firmly adherent Th1 cells that were used as positive control for the assay, 49.02±3% transmigrated, while 31.85±5.9% of the adherent iTreg transmigrated. In contrast, only 4.7±1.5% of the firmly adherent nTreg cells transmigrated, which was not significantly different from the transmigration observed in naive T cells (1.3±0.78%) (Figure 1B). Interestingly, re-activation of nTreg by different protocols as detailed in our discussion below did not change their adhesive or transmigration behavior on TNFα or IL-1β stimulated endothelial monolayers (data not shown).
Given the enhanced adhesive and transmigration behavior of both Th1 effector cells and Foxp3+ iTreg, we also sought to determine the molecules expressed by these cells that supported their interactions with cytokine activated endothelium. Specifically, we investigated the expression of chemokine receptors and sialyl-transferases involved in the synthesis of selectin ligands. As anticipated, we found that Th1 effector cells and Foxp3+ iTreg have different expression patterns of chemokine receptors. Notably, CCR5 was expressed by Th1 cells but not by iTreg, while CCR6, CCR8 and CCR9 were expressed by iTreg but not by Th1 cells (Figure 2A). CCR7 was also broadly expressed by Foxp3+ iTreg but only minimally expressed by Th1 cells. Furthermore, Foxp3+ iTreg and Th1 cells shared a high level of S1PR1 expression, which is required for lymphocyte egress from the lymph nodes en route to the blood and peripheral sites of inflammation (30). Foxp3+ iTreg also showed significantly more expression of α(1,3)-fucosyltransferases (FucT)-IV and less expression of β(1,6)-N-acetyl glucosaminyltransferase (Core2 or C2GnT) mRNA compared with Th1 cells, while the levels of FucT-IIV were comparable. These differences in glycosyl- transferase expression correlated with a marked increase of surface hyper-glycosylated CD43 in iTreg compared with Th1 (Figure 2B). Furthermore, the synthesis of functional selectin ligands, which is also glycosylation dependent, was very strong in iTreg and interactions with immobilized CD62E and CD62P under shear flow conditions were at levels comparable to those of Th1 (Figure 2C). Interestingly, the addition of retinoic acid to iTreg differentiation cultures not only enhanced the differentiation and viability of Foxp3+ iTreg, as suggested by others (31, 32), but also enhanced their ability to bind CD62P. In contrast, neither naïve CD4+ T cells nor nTreg showed measurable expression of CCR5, CCR6, CCR8 and CCR9 genes. We did find low level CCR7 expression in nTreg and high levels of S1PR1 in both naïve CD4+ T cells and nTreg. Consistent with their poor adhesive and transmigration behavior (Figure 1), neither naïve CD4+ T cells nor nTreg expressed detectable levels of hyper-glycosylated CD43 on their surface, and neither interacted with immobilized CD62E and CD62P under shear flow conditions (data not shown).
The robust adhesion of Foxp3+ iTreg to both TNFα activated endothelial cells and purified selectins led us to predict that iTreg would also have a regulatory influence on endothelial function. To examine this possibility we used two different in vitro systems to test the contact-dependent and contact-independent influences of iTreg on endothelial cells. The contact mediated influence of iTreg on endothelial cells was tested by first allowing iTreg to respond to endothelial antigen presentation and then evaluating their regulatory influence on endothelial activation by pro-inflammatory cytokines. The contact-independent effects of iTreg on endothelial function were evaluated by exposing endothelial monolayers to TNFα or IL-1β in the presence of supernatant taken from iTreg re-activated by endothelial antigen presentation or by TCR-complex cross linking. During preliminary studies for the contact inhibition experiments, we established that pretreatment with IFNγ caused MHEC to express I-Ab (Supplementary figure S3) and efficiently present antigen to stimulated Foxp3+ iTreg (shown below in Figure 4A) and Th1 cells (data not shown).
In order to determine the influence of iTreg contact on subsequent leukocyte-endothelial interactions, we added iTreg to TNFα stimulated MHEC monolayers and then measured Th1 cell adhesion to the MHEC under shear flow conditions. Contact with iTreg reduced the numbers of Th1 cells that accumulated on MHEC monolayers by 55% (from 75±0.5% to 33.5±9.5% of Th1 cells loaded) and also suppressed the transmigration of accumulated Th1 by 69% (from 23.5±0.5% to 7.45±0.55% of Th1 controls) (Figure 3A). Next, we measured the influence of iTreg contact on endothelial expression of surface selectins and found that iTreg suppressed the numbers of EC expressing CD62P by 85% in response to TNFα (from 64±3.8% to 9.6±2% CD62P+ MHEC) and by 90% in response to IL-1β (from 53.6±5.6% to 5.3±1% CD62P+ MHEC). Similarly, the number of MHEC expressing CD62E was reduced by 83% in response to TNFα (from 56±7.2% to 9.6±2% CD62E+ MHEC, P<0.01) and by 86% in response to IL-1β (from 52±4% to 7±4.7% CD62E+ MHEC) (Figure 3B). These experiments demonstrated that contact with activated iTreg suppresses cytokine mediated endothelial adhesiveness to Th1 cells and that this response is associated with reduced endothelial surface expression of E- and P-selectin. In both experiments, we found no evidence that iTreg contact damaged endothelial cells as there was no effect on the expression of I-Ab, H2-Kb, intercellular adhesion molecule 2 (ICAM-2;CD102), or the apoptosis marker Annexin-V after more than 8 hours of co-culture with iTreg (data not shown). In addition, iTreg differentiated without the addition of RA showed equal potency in contact induced suppression of EC adhesiveness to Th1 cells and selectin expression as compared with iTreg-RA (data not shown).
Both contact-dependent and contact independent mechanisms have been described for Treg suppression of T cell and dendritic cell function (33). We therefore tested if the in vitro influences of iTreg on endothelial function as described above could be duplicated by exposure of EC to supernatants from activated Treg. For this purpose, iTreg supernatant was collected from re-activated iTreg and levels of TGFβ1 were evaluated by ELISA to confirm activation. We found that Foxp3+ iTreg stimulated by either antigen-pulsed MHEC or plate bound anti-CD3ε readily secreted detectable amounts of TGFβ1 (335±31pg/mL and 513±61pg/mL, respectively) (Figure 4A and B). Interestingly, contact with MHEC alone, even without antigenic stimulus, induced TGFβ1 secretion from activated iTreg (62±16pg/mL) (Figure 4A), while contact with the culture dish did not (Figure 4B). Addition of iTreg supernatant to MHEC monolayers together with TNFα resulted in the suppression of Th1 cell accumulation on the MHEC monolayer by 65% (from 1080±192 to 378±60 Th1 cells/mm2) (Figure 4C). iTreg developed without RA secreted TGFβ1 at similar levels to iTreg-RA, and the supernatant collected from these iTreg was equally suppressive for EC functions as supernatant from iTreg-RA (data not shown).
It is well documented that prolonged conditioning of endothelial cells with TGFβ1 (12–24 hours) leaves endothelial cells refractory to stimulation by TNFα (34–36), although there is no published evidence to suggest that TGFβ1 can actively block TNFα stimulation of endothelium without extended preconditioning. Because we found that re-stimulated iTreg secrete high levels of TGFβ1, we tested whether TGFβ1 is the primary suppressive factor in Foxp3+ iTreg supernatant. Accordingly, we designed an experiment in which TGFβ1 instead of iTreg supernatant was added to MHEC during activation with TNFα (Figure 4C). Surprisingly, we found that TGFβ1 treatment given concurrently with TNFα suppressed MHEC adhesiveness and reduced the number of Th1 cells bound to the monolayer by 59% (from 930±101 to 384±127 Th1/mm2) (Figure 4D). This suppression was also associated with a significant reduction (~60–80%) in the number of selectin expressing EC (data not shown). These data show that the suppressive effects of iTreg and TGFβ1 on endothelial activation is rapid acting and does not require several hours of conditioning of endothelial cells as previously described (36).
In order to study test the suppressive effects of iTreg secretion products on endothelium, we collected supernatants from iTreg stimulated with plate bound anti-CD3ε. These supernatants contained TGFβ1 at comparable levels to supernatant taken from iTreg stimulated by MHEC antigen presentation (Figure 4A and B) and exerted a similar suppressive effect on MHEC selectin expression and Th1 adhesiveness (data not shown). In order to examine the role of TGFβ signaling in our model of iTreg suppression, we performed experiments with the ALK5 inhibitor SB431542, which blocks TGFβ signaling via the TGFβ type-I receptor (22, 23). Foxp3+ iTreg supernatant given to MHEC simultaneously with TNFα produced a 72% reduction in the ability of MHEC to support firm adhesion of Th1 cells under flow conditions (from 834±72 to 238±24 Th1/mm2), while pretreatment with the ALK5 inhibitor reversed this suppression to levels that were not significantly different from TNFα alone (Figure 5A). Similarly, iTreg supernatant caused a 70% reduction in selectin expression by MHEC in response to TNFα (from 100% to 31.7±5.5%), which was also completely reversed by pretreatment with the ALK5 inhibitor (109.3±0.85%, non-significant from TNFα alone) (Figure 5B). These data indicate that the suppressive effect of iTreg was primarily mediated by secretion of TGFβ1 and inhibitory signaling through ALK5, which activates the Smad2/3 phosphorylation cascade (37–39). Given the fast action of the ALK5 related Smad2/3 signaling cascade, these results could explain how iTreg interfere with the rapid translocation of NF-κB prompted by treatment with TNFα. Since ALK5/Smad2/3 activation has an obvious influence on transcriptional events, we further evaluated whether iTreg supernatant suppressed TNFα induced expression of selectin mRNA (Figure 5C). We found that iTreg supernatant suppressed TNFα mediated induction of CD62E mRNA by 89% (a decrease from 4.9±0.33 to 0.54±0.04 fold induction) and CD62P mRNA by 53% (a decrease from 6.7±0.2 to 3.12±0.1 fold induction). Addition of ALK5 inhibitor alone or in combination with TNFα, did not increase surface expression of endothelial selectin (data not shown). These data indicate that iTreg supernatant acts at the transcriptional level to suppress endothelial selectin expression.
To explore the in vivo significance of our findings we used two different models of inflammation. First, interactions of iTreg with microvascular endothelium was studied by intravital microscopy of the mouse cremaster muscle following intrasrotal injection of TNFα (27, 28). Adoptive transfer of fluorescently labeled iTreg into the femoral artery demonstrated that iTreg effectively migrate to post-capillary venules at the site of inflammation and exhibited robust rolling interactions relative to adoptively transferred naïve T cells (3.67±0.34 compared with 0.36±0.082 rolling cells/minute, respectively, n=35 vessels in 5 mice) (Figure-5A). Interestingly, the number of rolling iTreg per minute was comparable to that observed with adoptive transfer of Th1 effectors (2.44±0.35 rolling cells/minute, n=22 vessels in 4 mice) measured with identical activation conditions and experimental setup (P. Alcaide, unpublished observations). In order to determine whether iTreg secretion products could also suppress inflammation in vivo we used a model of TNFα induced acute peritonitis. We pre-treated mice (i.p.) with TGFβ1 or supernatant collected from reactivated iTreg-RA and then administered an i.p. injection of TNFα to induce peritonitis. Peritoneal lavage fluid was then collected after 4hr and leukocyte counts were performed (Figure-5B). Mice pretreated with iTreg-RA supernatant or TGFβ1 had a marked decrease in peritoneal leukocyte count (3.16±0.33×106 peritoneal leukocytes/mouse, n=12; and 1.65±0.33×106 peritoneal leukocytes/mouse, n=11, respectively), as compared to mice pretreated with control media alone (7.78±0.93×106 peritoneal leukocytes/mouse, n=9).
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 (10–14, 40), 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, 39, 42). 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, 44). 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, 51) 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 (Figure 4) 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.
We would like to thank Grigoriy Losyev of the BWH flow cytometry core, for his assistance with sorting Treg throughout the study.
1This work was supported by National Institute of Health grants P0HL36028 (N. Grabie, A.H. Lichtman, F.W. Luscinskas), HL087282 (A.H. Lichtman), Foundation Antonio Martín Escudero (E. Maganto-García), K99RO HL094706 (P. Alcaide) and 1K08HL086672 (K. J. Croce). G.G. is a recipient of a fellowship from the Sarnoff Cardiovascular Research Foundation.