How Lymphatic Endothelial Cells Destabilize Regulatory T Cells.

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 43, No. 2How Lymphatic Endothelial Cells Destabilize Regulatory T Cells Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBHow Lymphatic Endothelial Cells Destabilize Regulatory T Cells Qingkang Lyu and Klaus Ley Qingkang LyuQingkang Lyu Immunology Center of Georgia (Q.L., K.L.), Augusta University. Search for more papers by this author and Klaus LeyKlaus Ley Correspondence to: Klaus Ley, MD, Medical College of Georgia at Augusta University, Georgia Immunology Center, Laney Walker Blvd, Augusta, GA 30912. Email E-mail Address: [email protected] https://orcid.org/0000-0001-9339-3672 Immunology Center of Georgia (Q.L., K.L.), Augusta University. Department of Physiology (K.L.), Augusta University. Search for more papers by this author Originally published29 Dec 2022https://doi.org/10.1161/ATVBAHA.122.318849Arteriosclerosis, Thrombosis, and Vascular Biology. 2023;43:215–217is related toHypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and TraffickingOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: December 29, 2022: Ahead of Print In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Miyazaki et al1 investigated the role of calpain in regulatory T lymphocyte stability and trafficking under hypercholesterolemia. The authors demonstrated that lysophosphatidic acid (LPA) induced overexpression of calpain in lymphatic endothelial cells (LECs). Targeting the calpain system by genetic means or by inhibitors increased the production of TGF (transforming growth factor)-β and affected afferent lymphatic transport, which was sufficient to stabilize regulatory T cells (Tregs) and regulate Treg trafficking.See accompanying article on page e66The stability of Treg cells is characterized by maintained Foxp3 (forkhead box P3) and CD25 (IL2ra) expression. Tregs have immunosuppressive capacity, usually measured as suppression of effector T-cell proliferation. Under physiological conditions, Tregs are largely stable and suppress inflammatory immune responses.2 However, chronic inflammation can result in instability of Tregs. Treg dysfunction can also be induced by a lack of Treg survival factors like IL (interleukin)-2. During atherosclerosis, a chronic inflammatory disease of arteries, Tregs can lose the expression of CD25 and Foxp3, resulting in loss of suppressive function.3,4 Defective Tregs may gain effector characteristics. It has been proposed that they can transform into Th1 (T-helper), Th17, or Tfh (follicular helper T cell) cells,3,5,6 all of which are pathogenic in autoimmune diseases. Therefore, maintaining the stability of Treg is very important to prevent the development of chronic inflammatory diseases. Previous studies have shown that proinflammatory cytokines like IL-6 and IL-12 can prevent FoxP3 expression and cause Treg instability, whereas increasing IL-2, TGF-β or TCR (T-cell receptor) signaling stabilized FoxP3 and induced proliferation of Tregs.4 TGF-β is widely expressed by multiple tissues, including LECs. TGF-β signaling is known to have a protective role in atherosclerosis.7Calpains are a family of Ca2+-dependent, nonlysosomal proteases. Based on their distribution in tissues, calpains can be categorized as tissue-specific calpains and conventional (ubiquitous) calpains. Calpain 1 and calpain 2 are the best-characterized conventional calpains. Their activity is tightly regulated by their endogenous inhibitor, calpastatin. Calpain 1 and 2 regulate adhesion, migration, and lymphangiogenesis of LECs by controlling degradation and phosphorylation of eNOS (endothelial NOS).8 Activated calpains are able to selectively degrade intracellular proteins like IκB (inhibitor of κB), leading to activation of NFκB (nuclear factor κB), a proinflammatory transcription factor. Overexpression of calpain in ECs was observed in hyperglycemia and in the presence of high levels of lysophosphatidylcholine, both of which may contribute to endothelial dysfunction and hypertrophy. A previous paper from this research group showed that lysophosphatidylcholine is able to potentiate calpain expression in vascular endothelial cells.9 Therefore, it is reasonable to speculate that LPA may also enhance the calpain system in LEC. Calpain is involved in TGF-β signaling because calpain can degrade the adaptor molecule SMAD2 in human macrophages under hypoxia.10 Calpain activation also regulates both latent TGF-β1 activation and TGF-β1 protein synthesis.11To investigate the role of calpain in LEC (Figure), the authors analyzed the lysophospholipid composition in a hypercholesterolemic lymphatic environment and found that LPA accumulated in lymph nodes. Using lymphatic endothelial cell lines, the authors explored the impact of LPA on calpain activity. LPA increased calpain expression and activity. Moreover, calpain was found to be involved in regulating cell migration and lymphangiogenesis, but not mitotic activity. LECs interact with lymphocytes, which raised the question of what effect overactivated calpain in LECs may have on the differentiation of T-cell subsets. The authors co-cultured LECs and CD4+ T cells and found that calpain 1 (Capns1) knockdown in LECs expanded Foxp3+ Tregs and upregulated Treg-associated cytokines, like IL-10 and TGF-β1, in the presence of LPA. Mechanistically, both the Tgfb1 gene and TGF-β1 protein expression were upregulated in LPA-stimulated Capns1 knockdown LECs. Blocking TGFBR1 (TGF-beta receptor-1) on CD4+ T cells with antagonists like LY-364947, SB43152 or SB525334 reversed Treg expansion. This suggested that the combination of LPA and Capns1 silencing in LECs may stabilize Tregs through increasing TGF-β1 production. Because TGF-β1 production is mediated by multiple signaling pathways, ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and MAPK (mitogen-activated protein kinase) pathway were also studied. Activated calpain can proteolyze MEKK1 (mitogen-activated protein kinase kinase) and perturb its downstream pathway ERK and JNK, resulting in the reduction of TGF-β1.Download figureDownload PowerPointFigure. Proposed mechanism of Treg destabilization by hyoercholesterolemia. Hypercholesterolemia increases lysophospholipids (blue wedge) including lysophosphatidic acid (LPA), which inhibits CAST in lymphatic endothelial cells (LEC; top). Lower CAST leads to activation of calpains, which suppresses TGF (transforming growth factor)-β secretion, induces IL (interleukin)-18 secretion and VCAM-1 (vascular cell adhesion molecule) expression. Reduced TGF-β (small font) suppresses regulatory T cells (Tregs; green) and is proatherogenic. Inhibiting calpain activity by siRNA (small-interfering RNA) or inhibitors (bottom) restores TGF-β secretion (large font), stabilizes and expands Tregs (green) and is antiatherogenic. Red arrow means increase (upward) or decrease (downward).Furthermore, Miyazaki et al1 explored a possible causative relationship of calpain activation in atherosclerosis. The authors first showed that calpastatin expression was reduced in LECs in hypercholesterolemic mice. Then they introduced Cast (calpastatin), the gene encoding calpastatin, under the Lyve1 (lymphatic vessel endothelial-1) promoter into mice, which enhanced Cast expression in LECs and, presumably, in LYVE 1+ resident macrophages, although this was not tested. Because the Lyve1 promoter also controls gene expression in resident macrophages and possibly other hematopoietic cells, bone marrow transplantation was performed to test for a possible hematopoietic contribution. The result indicated that hematopoietic Cast neither affected atherosclerosis development nor Treg expansion or TGF-β1 production. Overexpression of Cast reduced lesion size in the aorta and M1 macrophage infiltration in the lesions, suggesting suppression of atherosclerosis development. Treatment of high-cholesterol diet–fed Ldlr−/− mice with a TGF-β receptor antagonist neutralized the protective role of overexpressed Cast. Next, the role of calpain in the stability of Tregs was tested in an in vivo experiment. Overexpression of Cast significantly expanded Tregs in blood of hypercholesterolemic mice. Meanwhile, TGF-β1 levels were upregulated in mesenteric LNs in CAST-overexpressing mice.Alteration of LEC function may affect lymphatics and lymphocyte trafficking. To investigate both, the authors labeled lymphocytes and found that the afferent lymphatic trafficking of CD4 lymphocytes and Tregs were decreased under hypercholesterolemia, whereas the decrease was reversed in CAST-overexpressing mice. DNA array experiments indicated that Vcam1 (vascular cell adhesion molecule) and Il18 genes were upregulated in hypercholesterolemic mice. The authors’ data suggest that upregulated VCAM-1 protein in LECs may significantly reduce lymphatic transfer of CD4 T cells and Treg cells. It is known that IL-18 can regulate VCAM-1 through NFκB- and PI3K (phosphoinositide 3-kinase)-dependent pathways.8,12 The authors explored the impact of calpain on NFκB. As expected, IL-18 increased VCAM-1 expression. This increased VCAM-1 was prevented by inhibiting calpain expression, which inhibited NFκB translocation by proteolyzing IκB. Increased VCAM-1 expression induced by calpain appears to be sufficient to limit lymphocyte trafficking.This study provides solid evidence that overactivated calpain in LECs under hypercholesterolemia is sufficient to destabilize Tregs. Activated calpain also restrains afferent lymphatic transportation of lymphocytes by controlling TGF-β1 and VCAM expression. Although calpains are widely expressed, the present study specifically focused on the critical role of calpain in atherosclerosis progression. As expected, hypercholesterolemic mice exhibited elevated levels of calpain, mainly induced by LPA. Interestingly, overexpressed calpain destabilized Tregs by inhibiting TGF-β1 production and contributed to altered lymphatic trafficking by inducing VCAM-1 expression. Mechanistically, calpain regulated ERK and JNK by proteolyzing MEKK1, thereby inhibiting TGF-β1 production. Furthermore, the authors also demonstrated that calpain activation enables upregulation of the IL-18/NFκB/VCAM-1 axis by inducing IκB degradation and thus regulating lymphocyte trafficking.The study has several limitations. Administration of inhibitors like LY-364947 will interfere systematically, not only locally, with TGF-β1 signaling. Calpains participate in many physiological activities, including apoptosis, cell adhesion, migration, and proliferation.13,14 Thus, systematically targeting calpain activity may cause unexpected side effects.Although the role of calpain in regulating lymphocyte stability and lymphatic trafficking has been well studied, some points are still worth to consider. Except for LPA and lysophosphatidylcholine, it is unclear whether other components in blood or lymphatic system drive calpain activation or overexpression during atherosclerosis. Does over-activation of calpain participate in Treg transformation to Th1, Th17, or Tfh cells? In addition to atherosclerosis, high lysophospholipid levels and immune dysfunction are also found in other diseases, such as obesity and diabetes, which raises the question of whether targeting the calpain system may have beneficial effects under these conditions as well.Taken together, the present findings demonstrate that targeting calpain can mitigate atherosclerosis progression by stabilizing Tregs and regulating afferent lymphatic transportation of lymphocytes. Although there is still a long way to develop therapeutic drugs for clinical use, this study suggests that new drugs selectively targeting calpain or Cast may be useful.Article InformationSources of FundingNational Institutes of Health grant R35 HL145241.Disclosures None.FootnotesFor Sources of Funding and Disclosures, see page 217.The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to: Klaus Ley, MD, Medical College of Georgia at Augusta University, Georgia Immunology Center, Laney Walker Blvd, Augusta, GA 30912. Email kley@augusta.eduReferences1. Miyazaki T, Taketomi Y, Higashi T, Ohtaki H, Takaki T, Ohnishi K, Hosonuma M, Kono N, Akasu R, Haraguchi S, et al. Hypercholesterolemic dysregulation of calpain in lymphatic endothelial cells interferes with regulatory T-cell stability and trafficking.Arterioscler Thromb Vasc Biol. 2023; 43:e66–e82. doi: 10.1161/ATVBAHA.122.317781LinkGoogle Scholar2. 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Arteriosclerosis, Thrombosis, and Vascular Biology. 2023;43:e66-e82 February 2023Vol 43, Issue 2 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/ATVBAHA.122.318849PMID: 36579643 Originally publishedDecember 29, 2022 Keywordsendothelial cellEditorialscalpainlysophosphatidic acidarteriosclerosishypercholesterolemiaPDF download Advertisement