Constitutive GITR Activation Reduces Atherosclerosis by Promoting Regulatory CD4+ T-Cell Responses—Brief ReportHighlights
Objective—Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4+ effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood.
Approach and Results—Low-density lipoprotein receptor–deficient mice (Ldlr−/−) were lethally irradiated and reconstituted with either bone marrow from B-cell–restricted Gitrl transgenic mice or from wild-type controls and fed a high-cholesterol diet for 11 weeks. Chimeric Ldlr−/− Gitrltg mice showed a profound increase in both CD4+ effector memory T cells and regulatory T cells in secondary lymphoid organs. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased Gitrl and Il-2 transcript levels. Atherosclerotic lesions of Ldlr−/− Gitrltg chimeras contained more total CD3+ T cells as well as Foxp3+ regulatory T cells overall, leading to significantly less severe atherosclerosis.
Conclusions—These data indicate that continuous GITR stimulation through B cell Gitrl acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4+ T cells.
Atherosclerosis is a chronic inflammatory disease of the arteries, characterized by both innate and adaptive immune responses.1 Important players in modulating these immune responses are costimulatory molecules. The costimulatory molecule glucocorticoid-induced tumor necrosis factor receptor family–related protein (GITR) is considered to be important in T-cell homeostasis.2–4
GITR is used as a marker for regulatory T cells (Tregs), a cell type well known for its protective role in atherosclerosis.5,6 However, GITR is also expressed on naïve T cells, and its expression becomes more prominent on activation. Additionally, antigen-presenting cells and mast cells express GITR, whereas its ligand (GITRL) is predominantly found on antigen-presenting cells and endothelial cells.7,8
Gitr−/− mice demonstrate protection in several disease models (eg, asthma, collagen-induced arthritis, spinal cord injury, and experimental colitis), which was predominantly associated with an impaired T-cell effector function.9 Accordingly, studies using agonistic antibodies suggested that GITR plays a proinflammatory role within the immune system through its costimulatory effects on T cells. Activation of GITR on Tregs in coculture with responder T cells promotes resistance to Treg-mediated suppression.7,8 However, Tregs from Gitr−/− mice exhibit a normal capacity to suppress T-cell proliferation.10 Hence, the exact function of GITR in T-cell biology of several diseases, including atherosclerosis, still remains unclear.
By using a transgenic (tg) mouse featuring B-cell–restricted overexpression of GITRL (Gitrltg),11 we aimed to unravel the effects of B-cell Gitrl-induced GITR activation on atherosclerosis.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
Gitrltg Chimeric Ldlr−/− Mice Display an Enhanced CD4+ Effector-Like and Treg-Type Phenotype
Ldlr−/− mice lethally irradiated and reconstituted with bone marrow (BM) isolated from either Gitrltg mice (Gitrl BM transplantation [BMT]) or wild-type littermates were fed a high-cholesterol diet for 11 weeks. At euthanasia, body weight (28.4±0.24 g versus 29.1±0.82 g) and basic hematologic parameters did not differ (Table I in the online-only Data Supplement).
The thymus showed equal fractions of double-positive (CD4+CD8+) or single-positive (CD4+ and/or CD8+) T cells in both chimeras. However, Gitrl BMT mice contained significantly higher absolute numbers of CD4+ T cells, whereas numbers of CD8+ and double-positive (CD4+CD8+) T cells did not differ (Figure 1A). Analysis of Forkhead box P3 (Foxp3) expression indicated that this increase in CD4+ T cells in the thymus of Gitrl BMT mice coincided with an enlarged Treg compartment (CD4+ CD25+ Foxp3+; Figure 1B). Furthermore, Gitrl BMT mice displayed an increase in thymic gene expression of Il2, Foxp3, and Ifnγ while no differences in Il4, Il10, and Tgfβ gene expression levels were present (Figure 1C), suggesting an increased thymic output of effector and regulatory CD4+ T cells.
In compliance with previously published results under normal conditions,11 Gitrl chimeric mice showed a systemic increase in frequency and absolute numbers of CD4+CD25+Foxp3+ Tregs in secondary lymphoid organs (Figure 1D; Figure I in the online-only Data Supplement). Accordingly, mRNA levels of Foxp3 were also significantly increased in these mice compared with controls (Figure 1E; Figure II in the online-only Data Supplement). Remarkably, the increase in absolute numbers of CD3+CD4+ T cells was confounded by an increase of activated CD3+CD4+Foxp3− cells (Figure 1F), reflected by a prominent increase in effector memory T cells (CD62L−CD44+) with no differences in the central memory T cell compartment (CD62L+CD44+; Figure 1G). This increase of CD4+ T cells with either a regulatory or an effector-like phenotype in Gitrl BMT mice did not develop at the cost of the naïve CD4+ population because absolute numbers of naïve CD4+ T cells were comparable to wild-type BMT mice. When analyzing CD8+ T-cell distribution in the lymph node, we observed neither a difference in total numbers nor in their naïve, effector, and memory-like phenotype (Figure 1H). These data are in compliance with previously published results under normal conditions.11 Quantitative polymerase chain reaction analysis revealed a significant increase of Ifnγ mRNA expression, while Il2, Il4, Il10, and Tgfβ expression was unaltered in lymph node (Figure 1E) and spleen (Figure II in the online-only Data Supplement) of Gitrl BMT mice.
Overall, these data indicate that GITR affects the numbers of both anti-inflammatory Tregs as well as CD4+ effector memory T cells of the proinflammatory type.
Activation of GITR Delays Atherosclerosis Development in Gitrl BMT Mice
We next examined the role of GITRL-induced GITR activation in atherosclerosis. Gitrl mRNA expression was significantly upregulated in the aorta of Gitrl BMT mice (Figure 2A). Compared with wild-type BMT mice, plaque formation in the aortic root was significantly reduced in Gitrl BMT mice after consumption of a high-cholesterol diet for 11 weeks (Figure 2B). Smaller plaque area was associated with an increase in CD3+ T cells, whereas lesional collagen and macrophage content did not differ (Figure 2C–2E). We found that the increase in CD3+ T cells involves a higher number of Foxp3+ cells in the aortic root of these mice (Figure 2F). Moreover, mRNA levels of Foxp3 were significantly increased in the aorta of Gitrl BMT mice as compared with the controls. Additionally, even though not significant, Il2 and Il10 mRNA levels were also increased, whereas Ifnγ transcript did not differ (Figure 2G). Of note, the differences in plaque formation were not a result of changes in systemic lipid metabolism, as indicated by similar levels of total plasma cholesterol, very low-density lipoprotein/low-density lipoprotein, and high-density lipoprotein between Ldlr−/− mice reconstituted with Gitrl versus wild-type BM (Table II in the online-only Data Supplement).
In conclusion, GITRL-induced GITR activation indeed activates both pro- and anti-inflammatory CD4+ T cells, but seems to favor the Treg—over the effector T cell—response in an experimental model of atherosclerosis.
The role of GITR on effector T cells versus Tregs continues to be subject of controversy, and it remains undefined how it influences chronic inflammation in vivo. Our results strongly suggest that in vivo GITR stimulation via its unique ligand simultaneously increases the numbers of both CD4+ effector T cells and Tregs. However, such GITR activation seems to favor the Treg response over a proinflammatory effector T-cell reaction, leading to limited progression of atherosclerosis.
The simultaneous accumulation of regulatory and effector CD4+ T cells in Gitrl BMT mice resulting in less lesion formation appears somehow contrary, especially because previous reports have suggested that GITR triggering on Tregs leads to abrogation of their suppressive capacity.8,12 However, Gitrl BMT mice do not develop organ inflammation or autoimmunity, suggesting that Tregs in our model are fully functional and can still maintain homeostasis. Additionally, the accumulation of both cell types seems a direct consequence of enhanced proliferation rather than conversion of naïve CD4+ T cells because the increase was not caused by a reduction of the naïve T-cell pool.
Recent in vivo studies revealed that concordant expansion of Tregs and effector T cells can be a direct consequence of responsiveness of Tregs to IL-2 produced by effector T cells.13 This is in agreement to our data showing augmented IL-2 expression in thymus and aorta of Gitrl BMT mice, which is most likely a direct effect, because GITR crosslinking with agonistic antibodies can induce IL-2 production through TNF receptor-associated factor-5–mediated NF-kB activation.14
Even though we found less atherosclerosis in Gitrl BMT mice, CD3+ and Foxp3+ T-cell numbers were increased in the plaque of those mice compared with the controls. Interestingly, others have shown that Gitrltg mice contained more Tregs with an activated phenotype, characterized as CD62Llow and CD103high.11 Because CD62L is required for high endothelial venule-dependent lymphocyte entry into the lymph nodes and CD103 is necessary for the homing and retention of cells at the sites of inflammation, these data suggest that Tregs in Gitrl BMT mice are possibly more prone to enter inflamed peripheral tissue and, thus, may accumulate in the atherosclerotic lesions of these mice.
In our study, we have induced GITR activation through overexpression of Gitrl on B cells. One of the caveats of this model is that we could only study B-cell–induced GITR activation and that we cannot address the activation of GITR mediated via GITRL on other antigen-presenting cells, such as dendritic cells, and macrophages, which may induce an even more profound GITR activation on T cells.
In summary, we have shown that GITR acts as a costimulatory molecule inducing proliferation of both effector memory CD4+ T cells and Tregs in Gitrl BMT mice. During atherosclerosis development, GITR triggering induced by B-cell Gitrl overexpression might induce IL-2 production, causing the proliferation of CD4+ effector memory T cells. Simultaneously, exogenous IL-2 released by proliferating CD4+ effector memory T cells could promote Treg expansion. These Tregs might display an activated phenotype, enabling them to enter inflamed tissues, such as atherosclerotic lesions.
We thank Suzanne Aarts and Myrthe den Toom for excellent technical assistance.
Sources of Funding
This research was supported by the Netherlands Cardiovascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development and the Royal Netherlands Academy of Sciences for the GENIUS project “Generating the best evidence-based pharmaceutical targets for atherosclerosis” (CVON2011-19). This work was supported by the Netherlands Organization for Scientific Research (NWO; VICI grant to E. Lutgens and VICI grant to C. Weber), the Netherlands Heart Foundation (Dr E. Dekker established investigator grant to E. Lutgens), the Deutsche Forschungsgemeinschaft (FOR809 to E. Lutgens/N. Gerdes; SFB 1054-B04 to E. Lutgens; SFB1123-A5 to E. Lutgens/N. Gerdes; SFB1123-Z1 to RTAM; INST 409/150–1 FUGG, LMU to CW/RTAM), the Landsteiner Foundation for Blood Transfusion Research (LSBR#1014), and the European Research Council (ERC consolidator grant 681493 to E. Lutgens).
This manuscript was sent to Kathryn Moore, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.116.307354/-/DC1.
- Nonstandard Abbreviations and Acronyms
- bone marrow transplantation
- forkhead box P3
- glucocorticoid-induced TNF receptor family-related protein (Ligand)
- regulatory T cells
- Received February 8, 2016.
- Accepted July 5, 2016.
- © 2016 American Heart Association, Inc.
- Ait-Oufella H,
- Salomon BL,
- Potteaux S,
- Robertson AK,
- Gourdy P,
- Zoll J,
- Merval R,
- Esposito B,
- Cohen JL,
- Fisson S,
- Flavell RA,
- Hansson GK,
- Klatzmann D,
- Tedgui A,
- Mallat Z
- Ronchetti S,
- Nocentini G,
- Riccardi C,
- Pandolfi PP
- van Olffen RW,
- Koning N,
- van Gisbergen KP,
- Wensveen FM,
- Hoek RM,
- Boon L,
- Hamann J,
- van Lier RA,
- Nolte MA
- Almeida AR,
- Zaragoza B,
- Freitas AA
- Esparza EM,
- Lindsten T,
- Stockhausen JM,
- Arch RH
Profound increase in both CD4+ effector memory T cells and Tregs in secondary lymphoid organs of chimeric Ldlr−/− Gitrltg mice
Significantly enhanced number of Tregs in the thymus and aorta of chimeric Ldlr−/− Gitrltg mice
Increased Gitrl and Il-2 transcript levels in the aorta of chimeric Ldlr−/− Gitrltg mice
More total CD3+ T cells and Foxp3+ Tregs in atherosclerotic lesions of chimeric Ldlr−/− Gitrltg mice
Significantly less severe atherosclerosis in chimeric Ldlr−/− Gitrltg mice